isolation and development of oil producing microorganisms including thraustochytrids...
TRANSCRIPT
Isolation and development of oil producing microorganisms including Thraustochytrids from Indian and Australian marine
biodiversity
by
Dilip Singh M.Sc. (Biotechnology)
Submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
Deakin University
October 2014
iii
TABLE OF CONTENTS
List of Publications x
Acknowledgements xi
Abbreviations xii
Abstract xvi
List of Figures xix
List of Tables xxii
1. Summary 1
1.1. Introduction 1
1.2. Thraustochytrids as potential feedstock for the biodiesel
production
2
1.3. Scope of thesis 3
1.4. Thesis outline 6
2. Introduction
2.1. Introduction
2.2. Oil producing microorganisms
2.3. Collection and isolation of oil producing microorganisms
2.4. Mechanism of oil production in oleaginous microorganisms
2.5. Thraustochytrids as feedstock for lipid production
2.5.1. Habit and habitat
2.5.2. Classification and phylogenetic history
2.5.3. Morphology and physiology
2.5.4. Isolation and identification
2.5.5. PUFA biosynthesis in Thraustochytrids and their physiological
role in human health
2.5.6. Lipid production in Thraustochytrids
2.5.6.1. Carbon metabolism during heterotrophic cultivation
2.5.6.2. Examples of cost-effective carbon sources for heterotrophic
cultivation
2.5.6.3. Application of growth modulators during heterotrophic
growth to accelerate lipid accumulation
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2.5.6.4. Nitrogen metabolism and lipid accumulation
2.5.6.5. Dissolved oxygen levels during reproduction and lipid
accumulation in heterotrophic cultivation
2.5.6.6. Role of physical parameters such as pH, salinity and
temperature during heterotrophic cultivation
2.5.6.7. FAME analysis of oil for biodiesel synthesis
2.5.6.8. Manipulation of the FAME profile
2.6. Thraustochytrids for carotenoid production
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3. Isolation, characterization and heterotrophic cultivation study of a new
oleaginous marine yeast DBTIOC-ML3
3.1. Introduction
3.2. Materials and methods
3.2.1. Reagents and chemicals
3.2.2. Sample collection and isolation of strain DBTIOC-ML3
3.2.3. Effect of different carbon and nitrogen sources on DCW, lipid
production and fatty acid profile for strain DBTIOC-ML3
3.2.4. Impact of different C/N ratios on DCW, lipid production and
fatty acid profile for strain DBTIOC-ML3
3.2.5. DCW, lipid production and FAME profile for growth of strain
DBTIOC-ML3 on non-detoxified liquid hydrolysate of wheat
straw or SH
3.2.6. Fermenter studies of strain DBTIOC-ML3 in a 2L continuous
stirred tank reactor
3.2.7. Enzymatic profiling of the strain DBTIOC-ML3
3.2.8. Molecular identification of strain DBTIOC-ML3
3.2.9. Sugar and inhibitor analysis of wheat straw NDLH with HPLC
3.2.10. Lipid extraction and FAME analysis
3.3. Results and discussion
3.3.1. Selection and identification of strain DBTIOC-ML3
3.3.2. Enzymatic profiling of the strain DBTIOC-ML3
3.3.3. DCW and lipid production for strain DBTIOC-ML3 using a
variety of carbon sources
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3.3.4. Effect of different nitrogen sources on DCW and lipid
production
3.3.5. Effect of increasing concentration of glucose or xylose on DCW
and lipid production
3.3.6. NDLH and SH as carbon sources for DCW and lipid production
3.3.7. Fatty acid composition of strain DBTIOC-ML3 under different
culture conditions
3.3.8. Growth profile of strain DBTIOC-ML3 using NDLH as the
carbon source
3.4. Conclusion
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4. Isolation and screening of Thraustochytrids from Indian and
Australian biodiversity for ω-3 fatty acids and biodiesel production
4.1. Introduction
4.2. Materials and methods
4.2.1. Reagents and chemicals
4.2.2. Site selection and sample collection
4.2.3. Isolation of Thraustochytrids from of Indian and Australian
marine samples
4.2.4. Strain cultivation and maintenance
4.2.5. Lipid extraction and FAME analysis
4.2.6. Genetic identification of Indian and Australian Thraustochytrid
strains
4.3. Results and discussion
4.3.1. Isolation of Thraustochytrid strains
4.3.2. Morphological study of Thraustochytrid strains
4.3.3. Screening of Indian and Australian Thraustochytrids for DCW,
lipid, biodiesel and ω-3 fatty acid production
4.3.3.1. DCW and lipid production
4.3.3.2. Fatty acid profiling of strains for screening of FAB and ω-3
fatty acids
4.3.3.3. Fatty acid profile as marker for chemotaxonomic grouping
of Indian and Australian Thraustochytrid strains
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4.3.4. Molecular phylogeny of Indian and Australian Thraustochytrid
strains
4.4. Conclusion
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5. Optimization of heterotrophic cultivation of Thraustochytrids for
DCW, lipid for biodiesel and ω-3 fatty acid production using selected
strains
5.1. Introduction
5.2. Material and methods
5.2.1. Reagents and chemicals
5.2.2. Screening of selected Indian Thraustochytrid strains on different
carbon and nitrogen sources
5.2.3. Effect of addition of calcium and magnesium ions on DCW and
lipid production in medium having varying concentration of
glycerol and sodium nitrate in DBTIOC-1 and DBTIOC-18
5.2.4. Growth profile of DBTIOC-1 and DBTIOC-18 under optimized
media composition
5.2.5. Glucose and glycerol estimation in media with HPLC
5.2.6. Lipid extraction and FAME analysis
5.3. Results and discussion
5.3.1. Screening of selected Indian Thraustochytrid strains on different
carbon and nitrogen sources
5.3.1.1. Effect of carbon sources on DCW and lipid production
5.3.1.2. Effect of carbon sources on FAB and DHA production
5.3.2. Effect of nitrogen sources on DCW, lipid, FAB and DHA
production in strain DBTIOC-1 and DBTIOC-18
5.3.3. Effect of increasing carbon and nitrogen supply on DCW, lipid,
FAB and DHA production in DBTIOC-1 and DBTIOC-18
5.3.4. Effect of calcium and magnesium ions and increasing carbon
supply on DCW, lipid, FAB and DHA production in DBTIOC-1
and DBTIOC-18
5.3.4.1. Effect of calcium and magnesium ions on DCW and lipid
production
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5.3.4.2. Effect of calcium and magnesium ions on FAB and DHA
production
5.3.5. Growth profile of strain DBTIOC-1 and DBTIOC-18 under
optimized media composition
5.4. Conclusion
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6. Effect of propyl gallate on the accumulation of saturated fatty acids, ω-
3 fatty acids and carotenoids in Thraustochytrids
6.1. Introduction
6.2. Material and methods
6.2.1. Reagents and chemicals
6.2.2. Culture maintenance and DCW production
6.2.3. Effect of different concentrations of propyl gallate (PG) on
growth profile, lipid accumulation, lipid composition and
carotenoid content
6.2.4. Effect of different C/N ratios on dry cell weight, lipid
accumulation, lipid composition and carotenoid content
6.2.5. Lipid extraction and FAME analysis
6.2.6. Carotenoid extraction and quantification
6.2.7. RP-HPLC for carotenoid analysis
6.3. Results and discussion
6.3.1. Effect of carbon sources and propyl gallate on dry cell weight,
lipid accumulation, lipid composition and carotenoid content of
Schizochytrium sp. S31
6.3.2. Effect of concentration of propyl gallate (PG) on growth profile,
lipid accumulation, lipid composition and carotenoid content of
Schizochytrium sp. S31
6.3.2.1. Effect on growth profile
6.3.2.2. Effect on lipid accumulation and lipid composition
6.3.2.3. Effect on carotenoid content
6.3.2.4. Propyl gallate effect
6.3.3. Effect of different C/N ratios, at optimized propyl gallate
concentration (0.03 %), on dry cell weight, lipid accumulation,
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lipid composition and carotenoid content of Schizochytrium sp.
S31
6.3.3.1. Effect of propyl gallate on dry cell weight
6.3.3.2. Effect of propyl gallate on lipid accumulation and
composition
6.3.3.3. Effect of propyl gallate on carotenoid content
6.4. Conclusion
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7. Study of cell disruption methods and use of response surface
methodology for optimization of astaxanthin extraction from
Thraustochytrids
7.1. Introduction
7.2. Materials and methods
7.2.1. Reagents and chemicals
7.2.2. Isolation and molecular identification
7.2.3. Culture maintenance, DCW production and carotenoid profiling
7.2.4. Study of growth profile and astaxanthin production
7.2.5. Effect of cell disruption methods on carotenoid yield
7.2.5.1. DMSO mediated extraction
7.2.5.2. Acid mediated cell disruption
7.2.5.3. Mechanical cell disruption
7.2.5.4. HPLC analysis of carotenoids
7.2.6. Response surface methodology design for extraction
optimization
7.3. Results and discussion
7.3.1. Isolation and molecular identification of strain
7.3.2. Carotenoid profiling of Thraustochytrium sp. S7
7.3.3. Study of growth profile and astaxanthin production in different
growth phases of Thraustochytrium sp. S7
7.3.4. Effect of chemical cell disruption on astaxanthin yield
7.3.5. Effect of mechanical cell disruption on astaxanthin yield
7.3.6. Response surface methodology for extraction optimization of
astaxanthin
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7.4. Conclusion 167
8. Conclusions and Future directions
8.1. Conclusions
8.2. Future directions
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Bibliography 174
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List of Publications
1. Singh, D, Barrow, CJ, Mathur, AS, Tuli, DK, Puri, M, (2015), Optimization of
zeaxanthin and b-carotene extraction from Chlorella saccharophila isolated from
new Zealand marine waters, (In press), Biocatalysis and Agricultural
Biotechnology
2. Gupta, A, Singh, D, Barrow, CJ & Puri, M (2013), 'Exploring potential use of
Australian thraustochytrids for the bioconversion of glycerol to ω-3 and
carotenoids production', Biochemical Engineering Joural, vol. 78, no. 0, pp. 11-7.
3. Singh, D, Puri, M, Wilkens, S, Mathur, AS, Tuli, DK & Barrow, CJ (2013),
'Characterization of a new zeaxanthin producing strain of Chlorella saccharophila
isolated from New Zealand marine waters', Bioresource Technology, vol. 143, no.
0, pp. 308-14.
4. Singh, D., Mathur, A. S., Tuli, D. K., Puri, M., & Barrow, C. J. (2014). Propyl
gallate and Butylated hydroxytoluene influences the accumulation of saturated
fatty acids, omega-3 fatty acid and carotenoids in Thraustochytrids under
comunication in Journal of Functional foods
5. Singh, D, Mathur, AS, Barrow, CJ, Puri, M, Tuli, DK (2014), ‘High cell density
cultivation of novel Aurantiochytrium sp. DBTIOC-18 and Schizochytrium sp.
DBTIOC-1 for concurrent production of biodiesel and docosahexaenoic acid’
submitted in Algal Research
Award and Conferences
Deakin India Research Initiative (DIRI), Australia to attend the 3rd International
conference on Algal DCW, Biofuels & Bioproducts, Toronto Canada from 15th June
2013 to 18th June 2013
2012 Annual conference IFM Deakin University, The Pier, Geelong, Victoria,
Australia
2nd DIRI symposium “Frontiers in Science” November 2011, Delhi, India
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Acknowledgements A research endeavour can never attain fruition without the optimal combination of
inspiration and perspiration of committed heads, hearts and hands.
First and foremost, I take this opportunity to express my heartfelt gratitude to the
almighty and my family for continuous support, blessing, protecting and guiding me
throughout this period. I could never have accomplished this without the faith I have
in the Almighty and instrumental support from the family.
I convey my sincere gratitude to Sh. Anand Kumar, Dr. R.K. Malhotra, former
directors of Indian Oil Research and Development Centre, India and Professor Peter
Hodgson Director Institute for Frontier Materials, Deakin University, Australia for
initiating Deakin India Research Program at Indian Oil Research and Development
Centre, India and selecting me for this revered program. I sincerely thanks to them for
inspiration, guidance and facilities provided for this scientific pursuit. My reverential
thank to Sh. B.P. Das, ED (I/c), R&D Centre for his constant encouragement.
I express my profound sense of reverence to my supervisors Dr. Deepak K. Tuli and
Dr. Anshu S. Mathur (at Indian Oil Research and Development Centre, India) and
Professor Colin J. Barrow and Dr. Munish Puri (at Deakin University Australia) for
their intuitive and incisive guidance, support, motivation and untiring help during the
course of my PhD. The scholarly supervision, constructive suggestions, keen interest
and instant troubleshooting has helped immensely in whetting my interest for
undertaking and accomplishing this research enterprise. They have given me enough
freedom during my research and guidance through difficult times. I am thankful to the
Almighty for giving me mentors like them.
I feel privileged to accord my sincere thanks to Sh. Ravi P. Gupta, Dr. Ravindra
Kumar, Dr. Ajay K. Sharma, Dr. Alok Satlewal and other members of the BERC for
their valuable help and support whenever a need arose. I was immensely benefited by
the inspiring and cheerful atmosphere that prevails at BERC.
I express my effusive thanks and regards to Dr. M.P. Singh, CRM Biotechnology for
encouragement and guidance during the initial phases of the work. Thanks are owed
xii
to Dr. Ravi Sahai, Dr. M. Upreti and Mrs. H Kaur for their cooperation, care and
concern.
I take this opportunity to express my gratitude Mrs. Ravneet Pahwa, Country Director
Deakin India and to all supporting staff of Deakin University especially Helen
Woodall, Gayathri Vedanarayanan, Anuradha Gupta, Pawan Solanki. This quote of
thanks would be incomplete without mentioning the name of Elizabeth Laidlaw, who
was instrumental to help me out whenever I encountered any lab related problem
during my stay at Deakin University, Australia.
I profoundly acknowledge and appreciate the help from Analytical Division, HR,
Materials, Instrumentation, IS and Projects deptt. for their help, support. I owe deep
sense of gratitude to Dr.A. K.Gupta and P.K.Gupta in HR dept. for actively helping
me out in various administrative issues or issues associated with fellowship during my
Ph.D. work.
I would like to give a quote of thanks to my colleagues and fellow lab mates in Indian
Oil Research and Development Centre especially Dr. Preeti Mehta Kakkar, Nisha
Singh, Tirath Raj. Special thanks to Adarsha Gupta, Avinesh Byreddy and Shailendra
Sonkar for isolation of 18SrRNA gene and gel run pertaining to Australian strains and
for making my stay at Deakin University pleasant with cherishing memories. My
sincere thanks to Jacqui for teaching me HPLC and GC, Reinu, Selvi, Shailendra and
other members of the lab at Deakin University for their help.
Finally words fail me to express my appreciation to my family members, especially
my mother and my sister for their blessings, inseparable support, prayers and love. To
conclude, I express my gratitude to my father late Sri Rajendra Singh and Almighty
for their celestial blessings and providing me the moral strength to lead the life against
all odds. I owe all my success to them.
DDilip….
xiii
Abbreviations
ATCC American type culture collection
ALA α-Linoleic acid
AA Arachidonic acid
Acetyl-CoA Acetyl co-enzyme
ATP Adenosine triphosphate
AMP Adenosine monophosphate
ACL ATP citrate lyase
ASTM American Society for Testing and Materials
ANOVA Analysis of variance
ACCase Acetyl coenzyme A carboxylase
AFDD Acriflavin dye detection α Alpha
BLAST Basic local alignment search tool
BHT Butylated hydroxytoluene
C/N Carbon/nitrogen ratio CMC Carboxymethylcellulose
CCD Central composite design
DNA Deoxyribonucleic acid
DHA Docosahexaenoic acid
DPA Docosapentaenoic acid
DCW Dry cell weight
DGAT Diacylglycerol acyltransferase
DO Dissolved oxygen
DMSO Dimethyl sulfoxide
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Δ Delta
EPA Eicosapentaenoic acid
ER Endoplasmic reticulum
EFBs Empty fruit bunches
FAB Fatty acid for biodiesel
FAMEs Fatty acid methyl esters
FACS Fluorescence-activated cell sorting
FAS Fatty acid synthase
FFAs Free fatty acids
FISH Fluorescence in situ hybridization
GHG Greenhouse gas
GLA ϒ- Linoleic acid
GC-FID Gas chromatography fluid ionization detection
HCl Hydrochloric acid
HMF Hydroxymethyl furfural
HPLC High performance liquid chromatography
LCPUFAs Long chain polyunsaturated fatty acids
LA Linoleic acid
LCB Lignocellulosic DCW
MUFAs Monounsaturated fatty acids
MACS Magnetic-activated cell sorting
Malonyl-CoA Malonyl co-enzyme
ME Malic enzyme
μm Micrometer
mM Millimolar
xv
μl Microlitre
μg Microgram
NADPH Nicotinamide adenine dinucleotide phosphate
NDLH Non-detoxified liquid hydrolysate
NCBI National Centre for Biotechnology Information
ὠ Ω
PBRs Photobioreactors
PUFAs Polyunsaturated fatty acids
PKS Polyketide synthase
PCR Polymerase chain reaction
PG Propyl gallate
rRNA Ribosomal ribonucleic acid
RP-HPLC Reverse phase high performance liquid chromatography
RSM Response surface methodology
SFA Saturated fatty acids SH Synthetic hydrolysate
STR Stirred tank reactor
TAGs Triacylglycerols
TFAs Total fatty acids
TCA Tri carboxylic acid cycle
UV Ultraviolet
xvi
Abstract Oils from marine microorganisms are a potential alternative to first generation
biofuels, due to several advantages over plant based vegetable oils, such as higher
productivity, shorter life span, better process control of the cultivation and relatively
easy scale up for industrial applications. In addition, oleaginous microorganisms can
utilize a variety of nutrient sources. Due to high DCW and lipid levels oleaginous
microorganisms such as Thraustochytrids can be exploited for large scale oil
production. Thraustochytrids are marine micro-heterotrophs, and produce ω-3 fatty
acids in large quantity. Most scientific research conducted on Thraustochytrids has
focused on the development of heterotrophic growth strategies for the production of
high value nutraceuticals such as ω-3 oils and carotenoids. However, Thraustochytrids
also accumulate significant amount of saturated fatty acids and monounsaturated fatty
acids and so are suitable feedstock in biodiesel production.
In this thesis, fast growing oleaginous microorganisms including the yeast
Rhodotorula sp. strain DBTIOC-ML3, and several strains of Thraustochytrids, were
isolated from Pichavaram mangroves, Tamilnadu, India. Aurantiochytrium sp.
DBTIOC-18 and Schizochytrium sp. DBTIOC-1 were isolated from mangroves in
Goa, India. The yeast strain Rhodotorula sp. DBTIOC-ML3 was found to tolerate
high concentrations of growth inhibitors, such as furfural, hydroxymethylfurfural and
acetic acid, that are present in the xylose rich non-detoxified liquid hydrolysate from
dilute acid pre-treated wheat straw, without compromising DCW and lipid production,
thus eliminating the need to detoxify the hydrolysates before heterotrophic growth.
This resulted in a substantial decrease in cost and energy associated with the
detoxification step. Several strains of Thraustochytrids were also isolated from
Australian marine sites including Barwon Heads, Victoria, Australia. Comparison of
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the fatty acid profile of Thraustochytrids from Indian and Australian marine sites
revealed a higher percentage of saturated and monounsaturated fatty acids in the
Indian Thraustochytrids, with higher percentages of ω-3 fatty acids in the Australian
Thraustochytrids. Indian Thraustochytrid strains were genetically identified and
screened for DCW and lipid production using several carbon sources. From the
collected Thraustochytrids two strains, Aurantiochytrium sp. DBTIOC-18 and
Schizochytrium sp. DBTIOC-1, were selected and culture conditions were optimized
to enhance their DCW and lipid productivity. The addition of calcium and magnesium
salts in the media promoted glycerol utilization at high carbon to nitrogen ratios,
resulting in a substantial rise in volumetric production of dry cell weight (51.43 gL-1,
48.52 gL-1), fatty acids suitable for biodiesel (16.01 gL-1, 16.5 gL-1) and
docosahexaenoic acid (13.31 gL-1, 5.37 gL-1), with strain Aurantiochytrium sp.
DBTIOC-18 and Schizochytrium sp. DBTIOC-1, respectively.
Addition of propyl gallate to the media resulted in increased DCW and lipid
production. An increased supply of carbon at an optimal concentration of propyl
gallate (0.03 % w/v) under nitrogen limiting condition resulted in significant
enhancement in lipid (24.87 gL-1), SFA (15.46 gL-1), DHA (5.19 gL-1) and astaxanthin
production (452.26 μgL-1). Production of high value co-products such as DHA and
astaxanthin together with fatty acids suitable for biodiesel may be using in offsetting
biodiesel production costs. Improving the economic viability through complete and
efficient extraction of carotenoids from Thraustochytrids remains an important target,
one in which the cell wall is a major impediment. Complete extraction of carotenoid
requires the cell wall to be disrupted by chemical or mechanical methods. The effects
of chemical and mechanical methods on cell disruption were investigated to
maximizing astaxanthin extraction yields. Ultrasonication was found to give
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maximize astaxanthin content and later response surface methodology was used to
optimize the ultrasonication process and maximize astaxanthin yield. Solvent/DCW
ratio (71.57), power (37.98 %), pulse (40 seconds) and time (10 min) were the
optimized conditions for maximum astaxanthin extraction (167.01 μgg-1).
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LIST OF FIGURES
Figure 2.1. Fatty acid biosynthesis pathway in oleginous microorganisms 15
Figure 2.2.
Figure 2.3.
Figure 2.4.
Figure 3.1.
Figure 3.2.
Figure 3.3.
Figure 3.4.
Figure 3.5.
Figure 3.6.
Figure 4.1.
Figure 4.2.
Figure 4.3.
Figure 4.4.
Figure 4.5.
Figure 4.6
(a) Classification and taxonomic nomenclature of Thraustochytrids (b)
Microscopic image (40X) of Thraustochytrids
(a) Standard FAS elongase/desaturase pathway (b) PKS pathway for
PUFA production in Thraustochytrids
Optimised FAME profile for biodiesel production
Phylogenetic tree of strain Rhodotorula sp. DBTIOC-ML3
Enzymatic profiling of cell bound enzymes of strain DBTIOC-ML3
Fatty acid composition of strain DBTIOC-ML3 cultivated on (a)
different carbon sources and (b) different nitrogen sources
Fatty acid composition of strain DBTIOC-ML3 on different C/N ratio
using (a) glucose or (b) xylose as carbon source and yeast extract as
nitrogen source
Growth profile of strain DBTIOC-ML3 on NDLH in 2L fermenter
HPLC profile of fresh NDLH media (inoculated with strain DBTIOC-
ML3) and depleted media (40 h old) in a 2 L fermenter
Site selection and sample collection from (a) Mandovi-Zuari
mangroves, Goa, India (b) Barwon Heads, Victoria, Australia
(a) Isolation method for Thraustochytrids (b) Peripheral colonization
of pine pollen by Thraustochytrids zoospores
Thraustochytrid strains from India (a-d) and Australia (e-h) marine
sites
Comparison of (a) DCW and (b) lipid production in Indian and
Australian Thraustochytrid strains
(a) Qualitative GC-FID of fatty acid profile of DBTIOC-18 compared
with Schizochytrium SR21, with the last two peaks showing the
presence of DHA and DPA (b) Fatty acid profiles of Indian and
Australian Thraustochytrid strains showing DHA presence in all the
strains
Dendrogram of Indian and Australian Thraustochytrid strains
representing similarity and dissimilarity among strains based on fatty
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21
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40
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Figure 4.7.
.
Figure 4.8.
Figure 5.1.
Figure 5.2.
Figure 5.3.
Figure 5.4.
Figure 5.5.
Figure 5.6.
Figure 6.1a.
Figure 6.1b.
Figure 6.2.
Figure 6.3.
Figure 6.4.
Figure 6.5.
Figure 6.6.
acid profiles
PCR amplification of 18SrRNA gene (a) Indian Thraustochytrid
strains (b) Australian Thraustochytrid strains
Phylogenetic tree of Thraustochytrids isolated from Indian and
Australian marine sites (triangle-Indian Thraustochytrids, circle-
Australian Thraustochytrids)
(a) DCW and (b) lipid production of Indian Thraustochytrid strains
cultivated on different carbon sources
FAB and DHA content of Indian Thraustochytrid strains cultivated on
different carbon sources
(a) DCW and (b) lipid production of DBTIOC-1 and DBTIOC-18
cultivated on different nitrogen sources
FAB and DHA content of DBTIOC-1 and DBTIOC-18 on cultivated
different nitrogen sources
Effect of Calcium carbonate and Magnesium sulphate on (a) DCW and
(b) lipid production in DBTIOC-1 and DBTIOC-18 cultures
Growth profile of strain (a) DBTIOC-18 and (b) DBTIOC-1 at
different growth intervals
Effect of propyl gallate on fatty acid profile of Schizochytrium sp.S31
Flasks showing the effect of propyl gallate (0.03 %) on biomass colour
in Schizochytrium sp.S31 cultures
Growth profile of Schizochytrium sp. S31 with glycerol under different
concentrations of propyl gallate (a) OD600nm (b) DCW (c) lipid content
(d) change in SFA/PUFA ratio (e) astaxanthin content at different time
interval
Microscopic images (40X) of the cultures fed with glycerol and
different concentration of propyl gallate
Mechanism of action of propyl gallate on pyruvate metabolism related
to Kreb’s cycle and transhydrogenase cycle
Proposed action on propyl gallate on polyunsaturated fatty acid
biosynthesis via desaturases/elongases pathway
Effect of C/N ratios with propyl gallate (0.03 %) on DCW and lipid
production in Schizochytrium sp. S31 astaxanthin content
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xxi
Figure 6.7.
Figure 7.1.
Figure 7.2.
Figure 7.3.
Figure 7.4.
Figure 7.5
Figure 7.6.
Figure 7.7.
Effect of C/N ratios with propyl gallate (0.03 %) on astaxanthin
content in Schizochytrium sp. S31
Phylogenetic relationship of Thraustochytrium sp. S7 with other
Thraustochytrids
Carotenoid profile of Thraustochytrium sp. S7
Growth profile and astaxanthin production over different growth phase
Effect of chemical cell disruption methods on astaxanthin yield
Effect of mechanical cell disruption methods on astaxanthin yield
(a-b) Response surface 3D plot showing the significant interaction of
solvent/DCW ratio, with power, pulse length on response i.e.
astaxanthin yield
Relationship between predicted and experimental response for
astaxanthin (μgg-1)
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165
166
xxii
LIST OF TABLES
Table 2.1.
Table 2.2.
Table 2.3.
Table 2.4.
Table 3.1.
Table 3.2.
Table 3.3.
Table 3.4.
Table 3.5.
Table 4.1.
Table. 5.1.
Table 5.2.
Table 5.3.
Table 5.4.
Table 6.1.
Table 6.2.
Table 7.1.
Table 7.2.
Table 7.3.
Oil content (>20 % of DCW) of oleaginous microalgae
Oil content (>20 % of DCW) in oleaginous fungi
DCW and lipid content in Thraustochytrids under different heterotrophic
growth conditions
Comparison of different properties of microbial biodiesel and ASTM
specification
DCW and lipid production on different carbon sources
DCW and lipid production on different nitrogen sources
Effect of C/N ratio on DCW and lipid production
Sugar and inhibitor concentration of NDLH of dilute acid pre-treated
wheat straw, analysed by HPLC
DCW and lipid production in semi-continuous culture
Lane number on agarose gel representing different Thraustochytrid
strains
FAB and DHA content of Indian Thraustochytrid strains cultivated on
different carbon sources
FAB and DHA production of DBTIOC-1 and DBTIOC-18 cultivated on
different nitrogen sources
DCW, lipid, FAB and DHA production in DBTIOC-1 and DBTIOC-18
cultivated on different C/N ratios
Effect of calcium carbonate and magnesium sulphate on FAB and DHA
production in DBTIOC-1 and DBTIOC-18
Effect of propyl gallate on DCW, lipid production and composition in
Schizochytrium sp. S31
Effect of different C/N ratios with propyl gallate (0.03 %) on DCW and
lipid yields and fatty acid ratios of SFA and PUFA
Range of different factors studied in response surface methodology
Astaxanthin yields from experimental and predicted methods (using
model equation) with different conditions obtained from central
composite design (CCD)
ANOVA analysis of the quadratic model and its statistical significance
11
12
26
39
58
61
63
64
71
92
106
109
112
118
129
140
160
161
162
1
CHAPTER.1
1. Summary
1.1. Introduction
Depleting conventional energy resources, rising fuel prices, alarming greenhouse gas (GHG)
levels in the atmosphere and the effects of global warming are among the many important
environmental issues that the global community must grapple with in the coming decades (Puri
et al. 2012a). Population explosion and increased per capita energy consumption in both
developed countries and emerging economies (such as India, China, Russia and Brazil) are major
driving forces behind this crisis. The world population is projected to grow from 6.8 billion in
2010 to 8-10 billion in 2030 ('International Data Base, world population summary' 2010).
Combustion of fossil fuels (e.g. coal, oil and gas) is the primary source of energy and currently
supplies 80% of the total global energy demand, which will cause a decline in petroleum reserves
at a rate of between 2% and 3% per year and these are predicted to vanish completely within the
next 100 years (Stephens et al. 2010). Researchers are focusing extensively on the development
of alternative sources of energy such as nuclear, solar, wind, hydro and biofuel. Biodiesel is one
of the most prominent biofuel and is prepared by the transesterification of triacylglycerols
(TAGs) into FAMEs. In spite of the favourable impacts that the commercialization of biodiesel
could provide, the cost of production of oil raw materials is a significant cost deterrent (Lin et al.
2011).
Oil can be obtained from plants, but energy and acreage are required for sufficient production of
oilseed crops, contributing negatively to the overall economics of biodiesel production from
agricultural plant crops. Apart from economics, diverting edible oil commodity to biodiesel
2
production may also endanger food security leading to food vs fuel debate, thus undermining the
sustainability of biodiesel. Therefore, exploring ways to reduce the high cost of biodiesel is an
important research theme, particularly for methods focused on lowering the cost of oil raw
material. The use of microbes as feedstock for biodiesel production could be advantageous over
agricultural crops due to their shorter life cycle, lower land usage, easier scale up and the lesser
impact of seasonal variations (Sharma et al. 2011). Oleaginous microorganisms such as algae,
yeast (Gupta et al. 2012b) and fungus have capability to accumulate high concentration of lipids
and can be exploited for competitive production of biodiesel. Microbial biofuels, therefore, have
the potential to replace petroleum fuels, but their realisation depends on technological and
economic advances (Chisti 2007).
The vast marine biodiversity has become a major ecological niche for the isolation of different
microorganisms producing bioactive metabolites. Researchers are exploring marine biodiversity
for the isolation of oil producing microorganisms. Oleaginous microorganisms such as
Nannochloropsis, Thraustochytrids, Mortierella, Rhodotorula, Yarrowia, Humicola and
Cryptococcus have been isolated from marine environments. These are fast growing
microorganisms and that can accumulate significant lipid quantities as their DCW (Chisti 2007;
Mutanda et al. 2011). Despite several advantages over first generation biodiesel, microalgae
derived biodiesel faces several key challenges such as process control, harvesting cost (in case of
autotrophic production in raceways or PBRs). These issues result in higher lipid production cost
from microalgal DCW thus reducing their potential for commercial production of biodiesel.
1.2. Thraustochytrids as potential feedstock for the biodiesel production
Thraustochytrids are marine heterotrophic, fast growing and high lipid producing microalgae
(accumulate up to 80 % of their DCW as oil). Microalgae, such as Thraustochytrids that produce
3
high value co-products along with lipids for biodiesel may help to bring down the cost of
production of biodiesel, thus increasing the probability of commercial production of microalgal
biodiesel. Thraustochytrids have been the subject of significant attention from the international
scientific community as they are rich source of ω-3 polyunsaturated fatty acids (PUFAs),
particularly DHA (Docosahexaenoic acid), which have nutraceutical and pharmaceutical
applications (Curtis et al. 2004; Linko et al. 1996). Omega-3 fatty acid comprises almost 50 % of
total oil in Thraustochytrid biomass, with the remaining oil being potentially useful as feedstock
for biodiesel production, thus subsidizing the production cost associated with biodiesel
production. Globally market size for ω-3 fatty acids is projected to reach $34.7 billion in 2016
with 6.4 % annual growth rate ('The Global Market for EPA/DHA Omega-3 Products' Packaged
Facts 2012). In addition to PUFA, Thraustochytrids are a rich source of carotenoids (Aki et al.
2003; Quilodrán et al. 2010), an important pigment and colorant used in the food and
aquaculture industries. The global carotenoid market is projected to reach $1.4 billion by 2018
(BCC 2011). Thus Thraustochytrid biomass could be used for concurrent production of biodiesel
along with DHA, EPA and other high value co-products such as carotenoid. However, the
development of this kind of heterotrophic cultivation strategy requires further research, including
new strain discovery, optimised heterotrophic cultivation strategies and the use of low cost
carbon sources.
1.3. Scope of thesis
a. Isolation of fast growing and efficient oil producing Thraustochytrids from marine
biodiversity is a first step in their application to biodiesel production. India and Australian both
have vast marine diversity with diverse flora and fauna. Indian and Australia coastlines have
4
potential marine biodiversity for the isolation of oil producing microorganisms. The coastline
comprises headlands, promontories, rocky shores, sandy spits, barrier beaches, open beaches,
embayment, estuaries, inlets, bays, marshy land, mangroves and offshore islands (Sanil Kumar et
al. 2006), with varying physio-chemical conditions.
b. Optimization of heterotrophic cultivation conditions for enhanced DCW and lipid production
is the second important step for economic production of biodiesel and ω-3 fatty acids from
Thraustochytrid biomass. Medium composition appears to have significant influence on DCW
and lipid production. Multiple culture parameters such as carbon source, nitrogen source, C/N
ratio, pH, salinity and temperature influence DCW production and lipid accumulation. Glucose
and yeast extract are well-established carbon and nitrogen sources, respectively, for ω-3 fatty
acid production. However, the use of costly nutrients will increase the production costs and so is
not practical for the production of low value and high volume products such as biodiesel.
Therefore there is scope to develop cost effective heterotrophic cultivation strategies using low
cost and widely available carbon and nitrogen sources for concurrent production of biodiesel and
other high value co-products such as DHA and carotenoids.
c. Application of growth modulators to increase DCW and lipid production has been studied in
Thraustochytrid heterotrophic cultivation, although there is little published in this area. Addition
of growth modulators such as Tween 80, malic acid and ethanol has resulted in increased DCW
and lipid production in some systems (Ren et al. 2009; Taoka et al. 2008). Antioxidants such as
propyl gallate (used in the food industry as a preservative) are reported to block pyruvate
transport into mitochondria (Eler et al. 2009) thus accumulating pyruvate in the cytoplasm
leading to enhanced acetyl-CoA and NADPH supply in lipid biosynthesis. Shift in flux towards
5
lipid biosynthesis and enhanced supply of these two metabolites will ensure higher lipid
accumulation (Ren et al. 2009). The presence of LC-PUFAs, particularly DHA, DPA and EPA,
in biodiesel will reduce oxidative stability, and so must be removed from the oil. This can be
achieved by either blocking the biosynthesis of LC-PUFAs in microorganisms or fractioning out
LC-PUFAs from the oil. Application of desaturase inhibitors such as propyl gallate could
potentially block the biosynthesis of LC-PUFAs. The literature suggests that complete inhibition
of DHA, DPA and other PUFA synthesis from the FAME profile of oleaginous microorganisms
is not achievable, since PUFA are key end products of the lipid cascade in these organisms
(Kobayashi et al. 2011; Matsuda et al. 2012).
d. Thraustochytrids are well documented for their ability to produce carotenoids. Different types
of carotenoids such as ß-carotene and xanthophyll have been isolated from Thraustochytrids (Aki
et al. 2003; Burja et al. 2006). Carotenoids are intracellular metabolites localized beneath the cell
membrane; therefore cell membrane disruption is necessary for effective extraction of
carotenoids from the cells. Cell disruption followed by solvent extraction is two key steps in the
downstream processing of carotenoids and these steps impact both the quality and yield. Cells
must be disrupted effectively, exposing the carotenoids to solvents and enabling solvents to
extract the intracellular carotenoids. Since cell wall composition varies from species to species
extraction procedures need to be optimized for the specific microorganism of interest, to
maximise yield. Improving the economic viability of through complete extraction of carotenoids
is important for commercial scale production.
6
1.4. Thesis outline
Literature on biodiesel, including classifications and the potential advantages of microbial
derived biodiesel are given in detail in chapter two. This chapter also reviews information on
oleaginous microorganisms and their isolation, together with mechanisms of oil production in
oleaginous microorganisms. This chapter also sheds light on different aspects of Thraustochytrid
research, such as classification, phylogenetic information, morphology and physiology, isolation,
pathways for PUFAs production and on the impact of heterotrophic cultivation parameters on
DCW and lipid production.
Isolation and characterization of novel oleaginous yeast, Rhodotorula sp. DBTIOC-ML3 strain,
is given in detail in chapter three, which is isolated from Pichavaram mangroves, in Tamilnadu
India. This new strain can utilize sugars present in non-detoxified liquid hydrolysate, without the
need for detoxification, and produce high levels of DCW and lipid from these sugars.
Total 33 Thraustochytrid strains were isolated from Indian and Australian marine biodiversity
(Chapter four). In India samples were collected from Zuari-Mandovi mangroves, in Goa near the
Arabian Sea. In Australia samples were collected from Barwon Heads, Victoria near the Tasman
Sea. Thraustochytrid diversity from both Indian and Australian marine sites was compared using
statistical tools including multivariate analysis. These strains were screened for the presence of
ω-3 fatty acids and oil potentially useful for biofuel.
7
Chapter five describes selection of the strains Aurantiochytrium sp. DBTIOC-18 and
Schizochytrium sp. DBTIOC-1 for DCW and lipid production. These two strains were obtained
through the screening described in Chapter 4. This chapter also describes heterotrophic
cultivation strategies for these two strains, applied to enhance DCW and lipid production rich in
ω-3 fatty acids and carotenoids, along with fatty acids suitable for biodiesel production.
Detailed investigation on effect of propyl gallate on growth of Schizochytrium sp. S31 for
biodiesel production, together with the production of the high value co-products DHA and
astaxanthin is mentioned in chapter six. The impact of different growth conditions, including
variation in carbon source, the use of Propyl gallate, and the C/N ratio, were studied in order to
maximize heterotrophic cultivation productivity.
Chapter seven describes evaluation of the carotenoid profile of Thraustochytrium sp. S7. The
impact of different chemical and mechanical cell disruption methods on the astaxanthin yield
was studied. Response surface methodology was used to maximize astaxanthin yield through
parameter optimisation.
Discussion on findings of this work and subsequent conclusions on this thesis work is
summarized in chapter eight with regard to using Thraustochytrid for concurrent production of
biodiesel, DHA and carotenoids.
8
CHAPTER.2
2. Introduction
2.1. Introduction
Biodiesel is a promising source of energy that can potentially substitute for fossil fuels and
mitigate against GHGs emissions. Biodiesel is of interest since it can be produced from
biodegradable, non-toxic, and renewable feedstock. Biodiesel is compatible with existing
infrastructure for fuel distribution network, transport and power generation. Replacement of
fossil fuel with biodiesel will not only help to meet the growing energy demand of these two
sectors but also help to reduce GHGs, since both these sectors are major contributors of GHGs
emission (Chisti 2007). Biodiesel can be used either in the pure form or as blends with petro-
diesel in automobiles without any major modifications. Being renewable makes biodiesel an eco-
friendly option with the potential to transform the current world economy from petroleum based
to biofuel based, if economic targets and demand could be achieved. Biodiesel could potentially
provide sustainable energy and energy security, coupled with economic prosperity for rural
societies (Bajpai et al. 2006).
Based on the feedstock, biodiesel can be classified as first generation, second generation, or third
generation. First generation biodiesel are commercially produced from edible crops (e.g.
soybean, canola oil, palm oil) but the viability of first generation biodiesel is questionable
because of conflict with food supply, raising a Food vs. Fuel debate (Ajanovic 2011). Second
generation biodiesel (Non edible crops e.g. pongamia oil, jatropha) utilises feedstock that doesn’t
compete with food. However, many of these feedstocks lack efficient digestivity, require higher
capital investment, and still need vast areas of land for commercial production. This puts
9
pressure on the water supply and limiting commercial production. Microbial derived DCW is
used for the production of third generation biodiesel. Third generation biodiesel overcomes many
of the limitations of first and second generation biofuel, primarily because of their high growth
rate and oil productivity, and limited requirement for land (Minowa et al. 1995). Microalgae
have a relatively short life cycle, low land usage, are easier scaled up and are not impacted by
seasonal variation (Lee et al. 2013). Oleaginous microalgae can be grown in brackish water, sea
water or waste water decreasing the cost of production.
2.2. Oil producing microorganisms
Many microorganisms across different kingdoms exhibit the ability to store lipid intracellularly.
Heterotrophic microorganisms such as yeast are extremely efficient in utilizing different classes
of sugars such as pentose and hexoses, and can be grown for oil production on low cost carbon
sources. Most microalgae are photosynthetic but have the ability to switch between different
modes of growth, that is, phototrophic, heterotrophic and mixotrophic thus depending on the
photosynthetic machinery and carbon source. For example, Chlorella and Scenedesmus can be
cultivated in either of the three modes, whilst other microalgae such as Schizochytrium are
exclusively heterotrophic. Microorganisms that accumulate more than 20% of their dry weight as
lipids are designated as oleaginous microorganisms (Ratledge 1991). Lipid content in many
microalgae such as Schizochytrium, Botryococcus, Nannochloropsis, Dunaleilla (Table 2.1a) and
fungi such as Rhodotorulla, Mortierella (Table 2.2b) exceeds more than 50 % of their DCW,
depending on various physiological parameters. Culture conditions, nutritional status and
environmental factors significantly affect lipid accumulation and composition. Nitrogen stress,
salinity stress and mixing are the physiological factors that enhance lipid accumulation in the cell
10
during heterotrophic cultivation (Mutanda et al. 2011), but at the expense of cell growth.
Microorganisms with high lipid content coupled with high DCW productivity are good
candidates for biodiesel production (Table 2.1a and 2.1b). Photosynthetic microalgae like
Chlorella, when grown under heterotrophic mode, have shown higher DCW and lipid content
compared to growth under their phototrophic modes (Rodolfi et al. 2009). The heterotrophic
mode offers increased flexibility in terms of light independence, easy scale up for industrial use,
negligible likelihood of contamination, high degrees of growth control and reduced harvesting
costs due to higher cell density. Heterotrophic cultivation is expensive both in terms of cost of
infrastructure and carbon source. However heterotrophic process producing the high value
products may enable to offset the higher cost associated with heterotrophic processes.
11
Table 2.1a. Oil content (>20 % of DCW) of oleaginous microalgae, modified from (Bollmeier et
al. 1989; Lam et al. 2012; Mutanda et al. 2011; Rodolfi et al. 2009)
1 F/M Fresh water/marine
Microalgae
Habitat (/F/M)1
DCW productivity
(gL-1 d-1)
Lipid content (% of DCW)
Amphora F/M - 15-51 Botryococcus F/M 0.02-0.03 20-75 Chaetoceros M 0.04-0.07 14.6-40
Chlamydomonas F - 18-33 Chlorella F 0.002-3.64 2-64
Chlorococcum F 0.28 19.3 Crypthecodinium F 10 20.0-51
Dunaliella M 0.12-0.34 6-71 Ellipsoidion M 0.17 27.4
Ettlia F 0.46 36-42 Euglena F 7.70 14.0–35
Haematococcus M 0.05–0.06 25.0 Hantzschia M - 50-66 Isochrysis M 0.08–1.60 7–40 Monodus F 0.08-0.19 13-52
Nannochloris M 0.04–0.51 20–56 Nannochloropsis M 0.09–1.43 12–53
Navicula F/M - 24-51 Neochloris F 0.03-0.63 7–65 Nitzschia F/M - 16–47
Ourococcus F - 27-50 Pavlova M 0.14-0.16 30.9-36
Phaeodactylum M 0.003–1.9 18.0–57 Porphyridium M 0.23–1.50 9.0–60 Scenedesmus F 0.004–0.74 11.0–55 Selenastrum F - 21-28 Skeletonema M 0.09 13.3–51 Thalassiosira M 0.08 16-24 Tetraselmis M 0.12–0.59 8.5–26
Schizochytrium M 0.4-13 40-83
12
Table 2.1b. Oil content (>20 % of DCW) of oleaginous yeasts, modified from (Xu et al. 2013;
Yousuf 2012)
Microorganisms
Habitat (/F/M)*
Lipid content (% of DCW)
Aspergillus F/M 57 Candida F/M 58
Cryptococcus F/M 58-65 Cunninghamella F 35 Entomophthora F/M 43
Fusarium F/M 34 Humicola F/M 75 Lipomyces M 64 Mortierella F/M 66-86 Pellicularia F/M 39 Rhodotorula F/M 72 Torulaspora M 40-50 Trichosporon F 45 Umbelopsis F/M 20-40 Waltomyces M 64
Yarrowia F/M 40-60
2.3. Collection and isolation of oil producing microorganisms
Collection and identification of fast growing, hyper lipid containing microorganisms is one of the
key milestones in successful commercial biodiesel production. Collection of oleaginous
microorganisms is influenced by several climatic and physio-chemical factors such as annual
rainfall, salinity, dissolved oxygen level, temperature, pH, macronutrient levels, and carbon
content of the selected habitat (Mutanda et al. 2011; Pereira et al. 2011). Since habitat conditions
affect the physiology of local microorganisms, physiological conditions of a habitat differ from
place to place, offering enormous opportunities for isolation of diverse groups of oleaginous
13
microorganisms with high DCW and lipid yields. These microorganisms can be collected from
various habitats such as fresh water, brackish and eutrophic ecosystem, marine, hyper saline
ecosystem (Doan et al. 2011; R.A. Andersen 2005). Various parameters such as pH and
temperature of the site should be measured during sample collection.
Different techniques are currently being employed for the successful isolation of oleaginous
microorganisms, which include conventional methods such as medium enrichment, single-cell
isolation by micropipette, serial dilutions, micromanipulation, atomized cell sprays, gravimetric
separation, and high throughput techniques like FACS and MACS (Brennan et al. 2010; Chang
et al. 2005; Rodolfi et al. 2009). Serial dilution and single cell isolation are the two most widely
used techniques for the isolation, whereby single cells are collected by micropipette and placed
into culture medium followed by streaking on agar plates having different combination of
antibiotics (Brahamsha 1996). The combination of antibiotics should be carefully selected based
on the targeted microorganisms to be isolated. The two above mentioned techniques are easy to
implement and effective only for small sample size. For large scale screening, FACS and MACS
(Han et al. 2005; Sinigalliano et al. 2009; Takahashi et al. 2004) are most frequently used. In
FACS, samples are serially diluted and inoculated in 96 well plate followed by the addition of
lipid staining fluorescent dyes such as nile red (Chen et al. 2009). Based on fluorescence, these
cells are sorted into high, moderate and low lipid containing cells. In MACS, samples are serially
diluted and labelled with magnetic nanoparticles based on the surface properties of targeted
microorganism, these cells are then passed under a magnetic field leading to separation of
microorganisms based on their surface magnetic properties.
14
2.4. Mechanism of oil production in oleaginous microorganisms
Genes involved in fatty acid biosynthesis (FAS I) in oleaginous microorganisms are similar to
those of higher plants which produce palmitic acid or stearic acid as the end product. Conversion
of acetyl-CoA to malonyl-CoA, catalysed by acetyl coenzyme A carboxylase (ACCase), is the
first major step in fatty acid biosynthesis (Fig 2.1) (Khozin-Goldberg et al. 2011). The key
regulatory steps in fatty acid biosynthesis of oleaginous microorganisms are similar to those of
higher plants. The continuous supply of acetyl-CoA (the precursor for fatty acid synthetase) and
NADPH (which act as a reducing agent in fatty acid biosynthesis) are the two major regulatory
steps in fatty acid assimilation (Ratledge 2004). The supply of acetyl-CoA in turn depends on the
level of citric acid present in cytosol and the activity of ATP citrate lyase. AMP deaminase plays
an important role in synthesis of citric acid in the TCA cycle. AMP deaminase is reported to be
up-regulated under nitrogen limitation, which ultimately stops metabolism of isocitrate in the
TCA cycle and enables mitochondria to expel accumulated citrate into the cytoplasm, leading to
enhanced rates of fatty acid synthesis (Ratledge 2004). This enzyme can be up-regulated, either
by genetic engineering or mutation for increased lipid production, resulting in high lipid content
and high DCW without inducing nitrogen limiting conditions.
Cytosolic levels of malate appears to be another major parameter for regulation of lipid
accumulation. Malic acid is considered to be a major supplier of NADPH (Ratledge et al. 2002;
Ren et al. 2009; Wynn et al. 1999). Malate reduces NADP into NADPH, which acts as a
reductant during condensation reactions of acetyl- CoA with malonyl-CoA (Fig 2.1). One mole
of C18 fatty acid is synthesized by 16 Moles of NADPH, since each 3-keto-fattyacyl group
requires 2 moles of NADPH for the reduction. Availability of NADPH in the cytoplasm can be
altered for enhancing fatty acid synthesis. The majority of the lipids in cells are stored as TAGs
15
in the lipid body. Chlamydomonas reinhardtii has been extensively studied for the isolation of
genes and the identification of proteins involved in TAG assembly (Moellering et al. 2010).
Diacylglycerol acyltransferase (DGAT) reportedly plays a major role in the final acylation of
diacylglycerol. Lower activity of DGAT is considered to be the foremost hurdle for TAGs
accumulation (Snyder et al. 2009).
Figure 2.1. Fatty acid biosynthesis pathway in oleginous microorganisms, modified from
(Ratledge 2004)
16
2.5. Thraustochytrids as feedstock for lipid production
2.5.1. Habit and Habitat
Thraustochytrids have a wide geographic distribution, with strains isolated from across the globe,
including the Antarctic region (Bahnweg et al. 1972), Argentina (Rosa et al. 2011), India
(Damare et al. 2008), Hong Kong (Fan et al. 2007), Japan (Takao et al. 2005), and Australia
(Gupta et al. 2013b; Lee Chang et al. 2012). Thraustochytrids are exclusively marine micro-
heterotrophs, ubiquitous in marine and estuarine environments in both tropical and sub-tropical
areas. Thraustochytrids are predominantly associated with the degrading vegetation in mangrove
swamps, marine sediments, littoral microalgae, and seaweeds. They are considered an integral
part of marine and mangrove ecosystems, where they act as decomposers and help to recycle
carbon, nitrogen and phosphorus. Researchers have isolated some strains from the gills of fish and
bronchioles of molluscs, but the nature of the relationship between Thraustochytrids and
invertebrates remains largely unexplored (Jones et al. 1983; Rabinowitz et al. 2006).
2.5.2. Classification and Phylogenetic history
Morphological and physiological properties were the two major factors for considering
Thraustochytrids as primitive fungi but later on aligned with heteroknot microalgae (Fig 2.2a)
(Cavalier-Smith et al. 1994) along with diatoms based on the phylogenetic relationship.
Molecular analysis of their 18s rRNA genes established that they are phylogenetically different
from oomycetes but close to labrinthulids and recently been assigned to the subclass
thraustochytride. Thraustochytrids are placed in the Kingdom Chromista or Stramenipila (also
called Stramenopila), alongside brown algae, diatoms, oomycetes and a variety of flagellates.
17
Most of the members of this kingdom are saprophytic such as Thraustochytrids, get nutrients from
dead organic matter while some are parasitic, like members of Labyrinthulids.
Figure 2.2. (a) Classification and taxonomic nomenclature of Thraustochytrids (b) Microscopic
image (40X) of Thraustochytrids
a.
b.
18
2.5.3. Morphology and physiology
Thraustochytrids are eukaryotic, unicellular microalgae characterized by the presence of thread
like outward projections called ectoplasmic net (EN). These ectoplasmic net are branched
extensions of plasma membrane arising from an organelle called sagenogenetosome or
bothrosome. These organelles are hypothesised to be remnants of golgi body. Ectoplasmic net not
only helps in the attachment to surfaces and absorption of nutrients from surrounding, but also in
the clumping of the cells. Thraustochytrium reproduces through zoospore formation (Fig 2.2b)
with vegetative cell transforms into zoosporangium having 5-10 zoospores at the time of
reproduction, whereas Schizochytrium reproduces through a binary fission type of cell division
(Bongiorni et al. 2005). Schizochytrium are smaller in diameter but are faster growing than
Thraustochytrids. Zoospores are strongly attracted to chemo attractants like pectin, gelation and
pine pollens. These zoospores are characterized by the presence of two flagella of unequal length.
Thraustochytrids grows well in medium containing sea water due to their obligatory requirement
of sodium ions for cell survival and this can’t be replaced with potassium (Shabala et al. 2009).
They are unable to synthesize lysine via the diaminopimelic acid pathway, but are able to utilize a
broad spectrum of sugars, nitrogen sources and are efficient in tolerating variation in pH and
salinity (Fan et al. 2002a). Most of the members of this family reach stationary phase between 7-
10 days, with prominent lipid bodies clearly visible in vegetative cells under the microscope.
2.5.4. Isolation and identification
S. Raghukumar from India, G. Bremer from the UK and D. Honda from Japan have extensively
studied methods for the isolation of Thraustochytrids from various natural habitats. Baiting
techniques and direct plating are two well established protocols for Thraustochytrid isolation
19
(Bremer 2000). Pine pollens, brine shrimp larvae and bird feathers are used as bait for the
isolation (Burja et al. 2006; Rosa et al. 2011). Sterilized bait is incubated with sample for 1-2
weeks at 20-300C. Once the colonization on the baits is observed under the microscope, they are
streaked on agar plates. These agar plates are supplemented with antibiotics such as penicillin (0.5
gL-1), streptomycin (0.5 gL-1), rifampicin (0.05 gL-1) and antifungal like amphotericin B and
nystatin (0.01 gL-1) (Jakobsen et al. 2007; Taoka et al. 2010; Wilkens et al. 2012), since there is a
possibility for the culture to get contaminated with bacteria and fungi. For the direct plating
method, samples are serially diluted or washed with antibiotic plus antifungal solution and
streaked on agar plates having suitable antibiotic and antifungals.
Cells of Thraustochytrids can be easily marked out under a microscope as they have the
characteristic uniform spherical shape, show sporangium formation and a spore release pattern. A
fluorescent dye (acriflavin) based staining technique (AFDD method) was developed for rapid
identification of Thraustochytrids (Raghukumar et al. 1993). Acriflavin upon reaction with
sulphated polysaccharides components of the cell wall gives a characteristic red colour ring-like
appearance at the periphery, with a green coloured nuclear wall at the centre, when visualized
under violet to blue light using a fluorescent microscope. This method has its own limitations and
cannot be applied to Thraustochytrids having a thin cell wall or zoospores without a cell wall.
Efforts have been made to develop molecular tools for rapid and accurate identification of
Thraustochytrids. PCR and FISH based techniques have been developed using Thraustochytrid
genomic information (Mo et al. 2002; Takao et al. 2007). In these techniques, specific probes
(fluorescent probes such as ThsFL1 in case of FISH) are designed to hybridize with the genomic
DNA of Thraustochytrids. Confirmation of identity is accomplished by 18s rDNA sequencing,
which establishes the phylogenetic status of the isolated species.
20
2.5.5. PUFA biosynthesis in Thraustochytrids and their physiological role in human health
Polyunsaturated fatty acids can be classified into two groups, ω-3 and ω-6 fatty acids, based on
the position of the first double bond on the carbon chain as numbered from the opposite end from
the carboxyl functional group. The type of PUFA pathway present in Thraustochytrids is highly
debatable, with some authors advocating the presence of only the polyketide synthase (PKS)
pathway (Hauvermale et al. 2006; Lippmeier et al. 2009). However, recent finding from
Matsuda and co-workers reveal the existence of both FAS desaturase/elongase and PKS PUFA
forming pathways in Thraustochytrium (Matsuda et al. 2012). However, it remains unclear
whether both of these pathways are functional for the production of DHA in Thraustochytrium.
Based on recently reported literature, it is probable that some Thraustochytrids have two distinct
pathways for PUFA synthesis. That is, both the PKS and the FAS desaturases/elongases
pathways operate for DHA production in some Schizochytrium (Hauvermale et al. 2006) and
Thraustochytrium species (Matsuda et al. 2012; Nagano et al. 2011b).
Palmitic acid and stearic acid act as precursors for long chain PUFA biosynthesis. In the
desaturase/elongase pathway, oleic acid can either, depending on the activity of Δ12/Δ15
desaturases, enter into the ὠ-3 series starting from α-Linoleic acid (ALA; C18:3) and ending at
docosahexaenoic acid (DHA; C22:6), or into the ὠ-6 series starting from linoleic acid (LA 18:2)
and ending at docosapentaenoic acid (DPA; C22:5) (Fig 2.3a). The overall pathway involves
three elongation steps and more than three desaturation steps required for conversion of LA/ALA
to DPA/DHA. These desaturases were isolated and characterized mostly from DHA rich
Thraustochytrium aureum ATCC 34304. These enzymes may be single function or multifunction
in nature; catalysing several steps simultaneously. Kang (2010) isolated and purified the novel
TaNE gene, which poses multifunctional Δ9, Δ6, Δ5 elongases and Δ5 desaturase activity (Kang
21
et al. 2010), while Lee et al. also isolated the novel multifunctional protein TaELO exhibiting
Δ9, Δ6, and Δ5-elongase activity and catalysing elongation of linoleic acid (LA, C18:2; n-6) and
α-linolenic acid (ALA, C18:3; n-3) to eicosadienoic acid (EDA, C20:2; n-6) and eicosatrienoic
acid (ETrA, C20:3; n-3), respectively (Lee et al. 2008). Researchers have isolated several novel
single functional desaturases/elongases, such as Δ5/Δ4 desaturases from Thraustochytrium
aureum ATCC34304 and these have been expressed in Pichia and Brassica to validate their
activity (Kang et al. 2008; Qiu et al. 2001).
Figure 2.3a. Standard FAS elongase/desaturase pathway in eukaryotic cells, modified from
(Gupta et al. 2012a)
22
Like the marine bacteria Shewanella, some members of the Thraustochytrid family, such as
Schizochytrium, are reported to have PKS like pathways for DHA synthesis (Fig 2.3b) (Metz et
al. 2009). It is believed that PUFA synthase genes were laterally transferred from bacteria to
Schizochytrium during their evolution. Unlike fungi, Schizochytrium has a multi-subunit PUFA
synthase complex, which consists of an acyl carrier protein, 3-ketoacyl synthase and malonyl
coenzyme-A acyltransferase (Nagano et al. 2011b). Three genes of the PUFA synthase complex
were isolated and expressed in E.coli resulting in accumulation of DHA (n-3) and DPA in the
cell, whereas disruption in these genes resulted in impaired synthesis of DHA and DPA in
Schizochytrium (Hauvermale et al. 2006; Huang et al. 2008; Huang et al. 2011; Lian et al. 2010;
Lippmeier et al. 2009). This pathway starts with the formation of acetyl CoA-ACP and malonyl
CoA-ACP esters from acetyl CoA and malonyl CoA, respectively (Fig 2.3b). Condensation by 3-
ketoacyl synthase results in synthesize of the first intermediumte, 3-ketobutyryl-ACP, with the
release of CO2. This intermediumte undergoes several intermediumry steps (reduction of keto
group to alcohol by 3-ketoacyl-ACP reductase, elimination of one water molecule from alcoholic
carbon by dehydrase creating double bond, addition of two hydrogen atoms on double bond by
enoyl reductase) to form a butyryl-ACP complex. Successive addition of malonyl CoA-ACP to
an acyl complex will increase the carbon chain length of the fatty acyl-ACP complex (Fig 2.3b).
Double bonds, which are created during elimination of water molecule in each cycle, can move
along the carbon chain length of fatty acyl-ACP complex depending on the activity of the
isomerase enzyme.
23
Figure 2.3b. PKS pathway2 for PUFA production in Thraustochytrids, modiefied (Gupta et al.
2012a)
These PUFA play an important role in reducing the risk of heart attack and other cardiovascular
diseases. DHA is an physiologically essential fatty acid for brain and eye development,
especially in infants, and is often added to infant formula (Gupta et al. 2012a). Biochemical
function of DHA in the central nervous system has been studied in detail. Deposition of DHA in
the membrane of neuron can be used as a marker to assess the functional status of neurons
related to maintaining vision and brain, particularly in infants (Guesnet et al. 2011; Yu et al.
2013). In adults DHA is reported to induce apoptosis in cancer cells, which can reduce the risk of
cancer (Skender et al. 2014). An optimal dose of DHA can be useful for preventing age related
2 ACC- acetyl Co-A carboxylase; KS-3-ketoacyl synthase; KR-3-ketoacyl-ACP reductase; DH- dehydrase; ER-enoyl reductase; I-isomerase
24
macular degeneration (Querques et al.). Regular intake of DHA is reported to decrease hardening
of blood vessels and reduce the risk of atherosclerosis (Chen et al. 2003). Long term
consumption of DHA is reported to reduce the risk of type-1 diabetes (Poudyal et al. 2013) and
non-alcoholic fatty liver disease (Scorletti et al. 2014). DHA has a broad spectrum of activity in
human physiology, and the World Health Organization (WHO) has recommended the daily
intake of 1 g per day DHA to reduce the chances of some of these diseases (Bailey et al. 2003).
2.5.6. Lipid production in Thraustochytrids
The efficiency with which Thraustochytrids produce oils is species dependent. Species such as
Schizochytrium are fast growing and can accumulate up to 80 % of DCW as lipids (Yaguchi et
al. 1997), while others, such as Thraustochytrium aureum ATCC 34304, are slow growing
(Bajpai et al. 1991). Heterotrophic cultivation parameters such as the source of carbon and
nitrogen, C/N ratio, pH, salinity, temperature and oxygen concentration influence DCW
production and lipid accumulation (Gao-Qiang Liu 2010).
2.5.6.1. Carbon metabolism during heterotrophic cultivation
Thraustochytrids are highly efficient at utilizing a broad spectrum of carbon sources and are
reported to produce up to 48 gL-1 of DCW with 73 % total lipid content (Table 2.3). Glucose is
the most frequently used carbon source in microbial heterotrophic cultivation because of its
simple structure and preferential uptake by microorganisms in the medium (Singh et al. 1996;
Singh et al. 1997). DCW production is reported to increase with increasing glucose
concentration in the medium, up to 10-12 % glucose, after which growth is restricted (Bowles et
al. 1999; Iida et al. 1996). When glycerol was used as a carbon source (9-12%) in place of
25
glucose, similar growth patterns were observed in Thraustochytrid heterotrophic cultivation (Chi
et al. 2007b; Yokochi et al. 1998). This drop in growth rate may be the result of excessive
acetate formation during heterotrophic cultivation, which negatively affects intracellular pH,
internal osmotic pressure and amino acid synthesis, thus resulting in low DCW. When excess
acetate was removed by conversion into acetyl-CoA in Schizochytrium culture, significant
increases in DCW and lipid content were observed (Yan et al. 2013). High glucose or glycerol
concentrations should be avoided in the initial phase of culture to avoid excessive accumulation
of acetate in the cell. Therefore fed batch cultures are designed in a way to maximize DCW
production with the intermittent addition of glucose. Yokochi and co-workers reported no
significant change in DCW or lipid content when glycerol was fed instead of glucose (Yokochi
et al. 1998). These results were in agreement with the findings of (Chi et al. 2007b). However,
Scott and co-workers reported an increase in DCW and lipid content in Thraustochytrium ONC-
T18 culture when fed with glycerol alone or a 50 % mix of glucose and glycerol (Scott et al.
2011). As a carbon source, glycerol appears to promote PUFA synthesis over SFA or MUFA
(Chi et al. 2007b; Scott et al. 2011; Yokochi et al. 1998). Increases in DCW and lipid content
reflect the tight regulation of enzymes involved in glycerol metabolism, enabling efficient
utilization of glycerol in the medium. Most of the glucose or glycerol is consumed within 48-72
h of culture; resulting in maximum DCW and lipid content at 72-96 h. Extended periods of
culture will force the cells to utilize lipid reserves for survival, resulting in low DCW and lipid
content, an outcome not desirable for biodiesel production. SFA appeared to be consumed first,
followed by PUFAs under carbon starvation condition (Scott et al. 2011). Therefore carbon
starvation should be avoided to achieve fatty acids profile suitable for biodiesel synthesis. To
avoid carbon starvation, addition of glucose or glycerol is recommended at intervals of 48 h, to
26
promote DCW and lipid production. In addition to glucose, other carbon sources such as crude
glycerol, maltose, sucrose, starch and linseed oil have been used for culture however, with the
exception of glycerol, significant decreases, or at best no change in DCW and lipid production,
were observed when supplemented with these alternative carbon sources (Gupta et al. 2012a).
Thraustochytrids are unable to metabolize disaccharides efficiently, resulting in poor growth and
lipid accumulation with traces of DHA (Wu et al. 2005). This indicates either poor uptake of
these sugars or lack of efficient enzymatic machinery to break these sugars into glucose before
entering into glycolysis.
Table 2.3. DCW and lipid content in Thraustochytrids under different heterotrophic cultivation
conditions
Strain Days Carbon source
Carbon source
(%)
DCW ( gL-1)
Lipid (% of DCW)
Reference
Thraustochytrium sp. 28210 5 Starch 2.5 8.6 18 (Singh et al. 1996) Schizochytrium limacinum
SR21 4 Glucose 12 48.0 77.5 (Yaguchi et al.
1997) Schizochytrium limacinum
SR21 5 Glycerol/
Glucose 9 38 50 (Yokochi et al.
1998) Schizochytrium mangrovei
G-13 4.5 Glucose 4 14 78 (Bowles et al.
1999) Thraustochytrium sp.
KK17-3 3 Glucose 4 7.1 19.9 (Huang et al. 2001)
Schizochytrium KH105 4 Glucose 10 11.5 50 (Aki et al. 2003) Thraustochytrium sp. ONC-
T18 3 Glucose 6 28.0 81.7 (Burja et al. 2006)
Thraustochytrid strain 12B 3 Glucose 8 31.0 57.8 (Perveen et al. 2006)
Schizochytrium KH105 4 Distillery waste/
Glucose
8 30.0 43.0 (Yamasaki et al. 2006)
Schizochytrium limacinum SR21
4 Potato broth/
Glucose
7 21.2 - (Chi et al. 2007a)
Schizochytrium mangrovei 4 Coconut 33 28 50 (Unagul et al.
27
Sk-02 water 2007) Schizochytrium limacinum
OUC88 5 Glucose 6 27 - (Song et al. 2007b)
Schizochytrium limacinum SR21
- Biodiesel derived glycerol
- 2.5-8 45-50 (Pyle et al. 2008a)
Schizochytrium sp. G13/2S 2 Glucose 15 63 25 (Ganuza et al. 2008)
Aurantiochytrium sp. T66 8 Glycerol 14.5 100 63 (Jakobsen et al. 2008a)
Aurantiochytrium mangrovei MP2
4 Glucose 9 25.4 - (Wong et al. 2008)
Schizochytrium limacinum SR21
7 Glycerol 10 37.9 - (Chi et al. 2009)
Aurantiochytrium strain BL10
7 Glucose 14 59 73 (Yang et al. 2010)
Schizochytrium limacinum OUC88
5 Glucose 9 25.92 - (Zhu et al. 2008)
Schizochytrium sp. 5 Glucose - 71 50 (Ren et al. 2010) Schizochytrium limacinum 14 Glycerol 11-12 58 64 (Jung et al. 2010) Schizochytrium limacinum
SR21 5 Sweet
sorghum juice
50 9.4 55-70 (Liang et al. 2010)
Thraustochytrium ONC-T18 5 Raw glycerol
6 31.6 37 (Scott et al. 2011)
Aurantiochytrium sp. KRS101
4 Glucose 6 50.2 44 (Hong et al. 2011)
Schizochytrium sp. HX-308 7 Glucose - 92.7 50.26 (Qu et al. 2011) Schizochytrium limacinum
SR21 - Biodiesel
derived glycerol
6 15 40 (Ethier et al. 2011)
Aurantiochytrium limacinum SR21
7 Glycerol 10 61.76 65.2 (Huang et al. 2012)
Aurantiochytrium sp. KRS101
4 Glucose 8 28 64 (Kim et al. 2013)
Aurantiochytrium sp. KRS101
7 Glucose 10 34.4 36.3 (Hong et al. 2013)
Aurantiochytrium mangrovei BL10
5 Glucose 15 44 22 (Chaung et al. 2012)
2.5.6.2. Examples of cost-effective carbon sources for heterotrophic cultivation
The type of carbon source, dosage and input costs are crucial parameters to be taken into account
in the design of heterotrophic cultivation protocols for the biodiesel industry. Low cost carbon
28
sources such as waste glycerol, potentially obtained from the biodiesel industry or fish
processing industry, sugar cane juice, molasses, beer residues, soybean cake, coconut water and
sweet sorghum juice have been investigated for lipid production, as low cost carbon sources to
improve commercial viability (Gupta et al. 2012a; Pyle et al. 2008b; Quilodrán et al. 2009).
Thraustochytrids are reported produce higher DCW and lipid production with alternative carbon
sources to glucose, such as coconut water, raw glycerol or beer residue (Scott et al. 2011; Unagul
et al. 2007). This increase in DCW and lipids can be attributed to the presence of other necessary
growth factors and trace elements in these low cost carbon sources. Quilodran (2009) reported as
much as 2-7 fold increases in DCW and lipid accumulation when liquid waste residues from beer
or potato chips manufacturing were used as the carbon source. An inverse relationship between
DCW and DHA productivity was also observed, while DCW and lipid productivity were found
to vary proportionally (Quilodrán et al. 2009). These results were in agreement with findings of
Unagul and co-workers (2006), who used coconut water as a carbon source instead of glucose
and observed significant improvement in DCW with little change in lipid content at
concentration of 33 % or 100 % coconut water (Unagul et al. 2006). However, DHA content was
found to vary inversely with DCW production. Other carbon sources such as sweet sorghum
juice and sugar cane juices have been used in Thraustochytrid cultures. DCW was reported either
comparable or less than that produced when using glucose, while lipid content was increased in
these medium (Liang et al. 2010). This difference in DCW production with the use of coconut
water or beer residue illustrates the important role of trace elements and other growth factors in
DCW production and lipid accumulation.
The ability of Thraustochytrids to utilize carbon sources from a broad spectrum of waste
materials makes them an attractive microbial system for biodiesel production, although
29
continuous supply of some of these materials at an industrial scale is not sustainable for large
scale biodiesel production. Thraustochytrids isolated from mangroves are reported to have the
necessary enzymatic machinery for degradation of complex sugars such as cellulose and
hemicellulose (Nagano et al. 2011a). When Aurantiochytrium sp. KRS101 was fed with
complex polysaccharides such as carboxymethylcellulose (CMC), cellobiose and palm oil empty
fruit bunches (EFBs), low DCW and lipid contents were observed along with comparable
amounts of DHA in the lipid profile (Hong et al. 2012). The presence of functional
carboxymethylcellulase or cellobiohydrolase enzymes presents the opportunity to use cellulosic
material as a carbon source in Thraustochytrid cultures. Encouraged from these findings, Hong
and co-workers (2012) applied enzymatic hydrolysate of alkali pre-treated palm oil EFB and
simultaneous saccharification and heterotrophic cultivation methodology to Aurantiochytrium sp.
KRS101 culture. They were able to produce high DCW (34.4 gL-1) and significant lipid content
(36.3 %) along with high DHA production (5.4 gL-1) at levels far greater than that recorded for
other microorganisms such as Rhodotorula, Tricosoporon and Yarrowia (Hong et al. 2012).
However, caution should be exercised while designing heterotrophic cultivation medium using
cellulose as carbon source since different strains have different physiological properties.
2.5.6.3. Application of growth modulators during heterotrophic cultivation to accelerate
lipid accumulation
Cell membrane permeability plays an important role in nutrient uptake during heterotrophic
cultivation. Increased permeability will result in enhanced nutrient uptake, leading to increased
DCW and lipid accumulation. Growth modulators such as Tween 80 have been used to alter cell
membrane permeability in Thraustochytrid cultures. Taoka and co-workers (2011) used Tween
30
80 as enhancer with glucose (as carbon source) in cultures of Thraustochytrium aureum ATCC
34304 and reported significant increases in DCW and lipid accumulation (Taoka et al. 2011).
Palmitic and oleic acid contents were significantly increased in the presence of Tween 80, while
DHA productivity remained unchanged. Increases in palmitic acid concentration can be
attributed to enhanced activity of the fatty acid synthetase enzymes, whereas the conversion of
the fatty acid moiety of Tween 80 into oleic acid via the desaturase/elongase pathway resulted in
increased levels of oleic acid. This report also indicates the functional existence of both fatty acid
pathways, that is desaturase/elongase and PKS, in Thraustochytrium aureum ATCC 34304
(Taoka et al. 2011).
Comparative flux of acetyl CoA between Kerb’s cycle and fatty acid synthesis determines the
DCW and lipid accumulation in the cell, as both pathways share a common substrate. Shifts in
this flux towards fatty acid synthesis will result in more lipid accumulation facilitated by
enhanced production of NADPH. Since the levels of acetyl CoA and NADPH play an integral
role in lipid biosynthesis, continuous supply of these two metabolites will enhance lipid
accumulation. When the medium was supplemented with ethanol and malic acid in the late
phases of Schizochytrium culture, a significant rise in DCW and lipid accumulation was
achieved. Ethanol provided an acetyl group, while malic acid acted to enhance NADPH
generation. Addition of ethanol had little effect on DHA accumulation, as measured by FAME
analysis, but DHA levels increased following addition of malic acid into the medium (Ren et al.
2009), indicating the importance of NADPH in DHA production. Reports suggest that addition
of reducing agents such as sodium thiosulfate or methyl viologen can increase NADPH pools,
inactivate citrate synthase and activate the glyoxalate shunt that was not functional under glucose
(Feng et al. 2005; Mandal et al. 2009). All of the aforementioned changes appear to favour lipid
31
accumulation over the Kreb’s cycle. Sodium thiosulfate is known to exhibit oxygen scavenging
activity, thus preventing lipid degradation. When Ngangkham (2012) added sodium thiosulfate
in the mixotrophic culture of Chlorella sorokiniana, significant increases in DCW and lipid
content were observed. Additionally, SFA and MUFA content in the culture substantially
increased, while PUFA levels declined (Ngangkham et al. 2012).
2.5.6.4. Nitrogen metabolism and lipid accumulation
The availability and type of nitrogen sources in the medium are key parameters affecting total
lipid yield and fatty acid profiles. Nitrogen content dictates the cell division rate since most of
the vital processes of cell division, such as protein synthesis and nucleic acid synthesis, are
directly controlled by nitrogen levels in the medium. Generally, nitrogen is consumed in the
medium within 48-72 h. Once nitrogen is consumed, AMP deaminase is up regulated, shifting
acetyl CoA flux towards lipid biosynthesis. Glucose content under nitrogen stress, therefore,
significantly affects lipid production, with higher glucose levels resulting in higher lipid content,
due to perpetually more glucose being channelled into the fatty acid synthesis pathways (Bailey
et al. 2003). This is typical of oleaginous microorganisms. However, Ratledge and Wynn (2002)
proposed accumulation of lipids during the growth phase in Thraustochytrids. This suggestion is
supported by the findings of Ganuza and co-workers (2007) (Ganuza et al. 2008; Ratledge et al.
2002) who studied the growth of Schizochytrium sp. G13/2S with different carbon sources under
a fed-batch system. Lipid accumulation normally begins in the late log phase and continues until
the early stationary phase or until complete exhaustion of the carbon source. Prolonged
heterotrophic cultivation will result in the utilization of lipid reserves, causing a decrease in lipid
and DCW. To avoid this, DCW should either be harvested in the early stationary phase (between
32
4-5 days) or additional carbon source should be added to the medium (fed-batch culture). This
explains why productivity is higher in fed-batch cultures than in batch cultures with equivalent
quantities of carbon source. Nitrogen stress coupled with oxygen or phosphate limitations
resulted in increases in lipid content, although PUFA levels reduced significantly in the FAME
profile, indicating the importance of oxygen and phosphorus in lipid biosynthesis (Jakobsen et
al. 2008a). It was proposed that phosphate limited conditions enhance glucose 6-phosphtae
dehydrogenase activity in the early phase of culture, while malic enzyme activity increases in the
later phase of culture and isocitrate dehydrogenase activity remains lower during growth under
phosphate limitation (Ren et al. 2013b). These three enzymes play an important role in acetyl
Co-A and NADPH supply during lipid biosynthesis. Nitrogen depletion in the medium
reportedly enhances lipid content by several folds, but at the expense of DCW yield, so
appropriate C/N ratios need to be selected in the medium for optimal DCW and lipid production.
Thraustochytrium show a low optimal C/N ratio of about 10.4 (Kimura et al. 1999) as compared
to Schizochytrium at about 15.1 (Ratledge 2004). Higher C/N ratios result in higher lipid
accumulation at the expense of DCW yield. Researchers are attempting to develop two-step
culture methods to enhance DCW yield. Firstly, Thraustochytrids can be grown on high carbon
and nitrogen containing medium to enhance cell density, followed by subsequent transfer into
nitrogen depleted medium with high carbon content in the second step to increase cell weight
and lipid content (Bailey et al. 2003). Most Thraustochytrids are reported to grow well on
organic carbon sources such as peptone, tryptone, yeast extract, and corn steep liquor or
inorganic nitrogen sources such as ammonium sulphate, ammonium acetates, nitrates, and
monosodium glutamate (Barclay 1994a, 1994b; William et al. 2005). Ammonium acetate and
nitrates are reportedly a preferable inorganic nitrogen source in Thraustochytrid cultures.
33
Yokochi and co-workers reported high DCW when they fed ammonium acetate instead of yeast
extract (Yokochi et al. 1998), although lipid content remained unchanged. Similar trends were
observed by Huang et al (2001) and Chatdumrong et al (2007), where ammonium nitrate was
used as the nitrogen source (Chatdumrong et al. 2007; Huang et al. 2001).
Although the quantity of nitrogen sources needed for heterotrophic cultivation is far less than
that of carbon sources, the use of inexpensive nitrogen sources is still required to reduce costs
associated with biodiesel production. Inorganic nitrogen sources are significantly low cost than
their organic counterparts, but questions as to their longer term sustainability remains. The
ability of Thraustochytrids to utilize a wide range of nitrogen sources provides ample
opportunity to take advantage of potential inexpensive nitrogen sources in heterotrophic
cultivation. Researchers have grown Thraustochytrids on various low cost nitrogen sources such
as soybean meal, skimmed milk and distillery wastewater. Schizochytrium has been grown on
these nitrogen sources and substantial DCW and lipid accumulation was observed (Chatdumrong
et al. 2007; Yamasaki et al. 2006). However mixtures of nitrogen sources, such as peptone with
soybean meal, resulted in higher lipid content, but DCW production was reported to remain
constant (Chatdumrong et al. 2007), suggesting that optimised mixtures of inexpensive nitrogen
sources may be used without compromising DCW or lipid yield. Wastewater effluents from
tanneries, abattoirs, and the dairy industry, as well as waste materials such as sludge or landfill
leachate, containing nitrogen up to 4-5 gL-1, represent low cost but abundant nitrogen sources.
While these can be explored as alternative organic nitrogen sources (Carrera et al. 2003), they
must be pre-treated before use to remove toxic materials, as these can inhibit growth. The
balance between the cost of remediation and productivity must be analysed to establish the
suitability of these effluents as nitrogen sources.
34
2.5.6.5. Dissolved oxygen levels during reproduction and lipid accumulation in
heterotrophic cultivation
Dissolved oxygen levels in the medium are reported to have significant effects on cell density,
DCW production, lipid yield and FAME profiles (Qu et al. 2013). Thraustochytrids appear to
follow the typical two-stage growth strategy of oleaginous microalgae. The first stage is marked
by higher growth activities such as protein synthesis, DNA synthesis, resulting in increased cell
density with little accumulation of lipids. Most of the anabolic activities during the growth phase
are oxygen dependant, and thus they require more oxygen to achieve higher cell density, a key
criterion for increased lipid production. When the dissolved oxygen level was changed from 10
% to 50 % in a Schizochytrium culture, cell density increased by almost 4.5 times in 24 h, but
later dropped significantly at the mid culture stage (Chi et al. 2009). This can be explained in
terms of specific oxygen uptake rate, which is reported to be higher between 8-10 h, followed by
a decline at 40 h. This sheds some light on the role of dissolved oxygen in cell reproduction and
lipid accumulation (Chi et al. 2009). Zoospore production and zoospore-vegetative cell
transformation processes require large amounts of oxygen during the growth phase, which
subsequently decrease at the onset of lipid accumulation. Higher dissolved oxygen
concentrations appear to have a negative impact on DCW production and lipid accumulation in
Schizochytrium, whereas low DO concentrations favours higher DCW and lipid accumulation.
Bailey et al. (2003) reported an almost 6% increase in lipid content when DO concentrations
were altered from 40 % to 5 %. Subsequently, analogous results were obtained by Qu et al.
(2010), who demonstrated a significant increase in lipid content when the oxygen transfer
coefficient was changed from 150.1/h to 88.5/h (Bailey et al. 2003; Qu et al. 2011).
35
Low oxygen levels slow cell respiration and channel carbon resources into fatty acid
biosynthesis pathways. It should however be remembered that low oxygen levels with adequate
nitrogen supply will result in comparatively low DCW and lipid content with significantly
elevated DHA content in Aurantiochytrium culture. However coupled with nitrogen limitation,
low oxygen levels in the late log phase will induce DCW and lipid accumulation with increases
in SFA and MUFA. This observation by Jakobsen and co-workers underlines the role of
dissolved oxygen in PUFA biosynthesis via an oxygen independent, PKS pathway in
Schizochytrium. Sacrifice of cell density is not advisable for higher DCW and lipid production.
Therefore, a two-step aeration strategy has to be utilised for enhanced DCW and lipid yields. In
the first stage, oxygen levels should be kept high in lag to mid log phase (from 8h-40 h) of
growth to achieve adequate cell density, followed by a drop in oxygen levels, inducing lipid
accumulation and resulting in higher cell weight and lipid yield in the Schizochytrium culture
(Chi et al. 2009; Jakobsen et al. 2008b; Qu et al. 2011; Ren et al. 2010). However, contrasting
results were obtained by Ge et al. (2011) during culture of Thraustochytrium aureum ATCC
34304, who reported better DCW and PUFA yield when the culture was shifted from lower to
higher aeration at a particular pH (Yang et al. 2011); the opposite result to a reduction in lipid
yield observed for Schizochytrium. They also reported an increase in DHA productivity and yield
under high aeration. This contrasting behaviour of Thraustochytrids can be attributed to the
existence of a different functional PUFA biosynthesis pathway in Thraustochytrium aureum
ATCC 34304, that is, an oxygen dependent desaturases/elongases pathway. Under high aeration
conditions desaturase remains functional, catalysing conversion of SFA and MUFA to PUFAS
and ultimately to DHA/DPA. The species of Thraustochytrid therefore needs to be taken into
account while aeration strategy for high DCW and lipid production are being designed.
36
2.5.6.6. Role of physical parameters such as pH, salinity and temperature during
heterotrophic cultivation
Thraustochytrids are able to grow in a wide range of pH conditions, ranging from 4-9 (Arafiles
KHV et al. 2011; Kumon et al. 2002; Lee Chang et al. 2011; Perveen et al. 2006; Singh et al.
1996), while some reports suggest that some of the Thraustochytrids such as Ulkenia sp. will not
grow at highly acidic or basic pH. High DCW yield was observed at proximity to neutral pH
levels (Kumon et al. 2005; Kumon et al. 2006; Kumon et al. 2003), but final pH became alkaline
in some of the studies (Singh et al. 1996). This increase in pH is attributed to changes in the
medium composition at the end of cultivation. However, in some cases final pH fell into acidic
range, such as when Schizochytrium was cultured under high aeration. Formation of organic
acids in the late phase of the culture contributed to a drop in pH. No significant change was
observed either for lipid content or FAME profile except for increases in DHA content at higher
pH (7-8) (Perveen et al. 2006). However extreme, acidic or basic conditions can result in poor
growth, since these extreme conditions will not only affect the osmotic balance in the cell but
also generation of NADPH, which is a significant driver of lipid accumulation. Low pH (<5.5)
under high aeration resulted in substantial improvements in DCW and DHA content in
Thraustochytrium culture (Yang et al. 2011). Acidic conditions facilitate the uptake of nutrients
from the medium and increase the supply of NADPH, whereas high aeration will ensure a
continuous supply of oxygen required for the desaturase enzyme. Enzyme associated fatty acid
biosynthesis requires several elements, such as manganese, magnesium, sodium, potassium,
cobalt as co-factor. A supply of these elements in the medium is mandatory for enzyme action.
Sodium ions must be supplied to the medium for Thraustochytrid culture, since these facilitates
37
phosphate uptake, promote growth and cannot be replaced with potassium (Garrill et al. 1992).
Therefore the medium should be supplied with either natural or artificial sea water for these
elements. However, several Schizochytrium strains are reported to grow in zero salinity, but
DCW and lipid content were significantly reduced, confirming the need of these elements in the
medium. Yokochi et al. reported linearly proportional increase in DCW yield with sea water
strength up to 50 % (36 gL-1 sea salt in water mimic 100 % sea water) (Yokochi et al. 1998).
Many researchers have recommended the usable limit of salinity between 15 %-25 %, or in some
cases as high as 50 %. Strains isolated from mangroves and estuaries exhibit superior salinity
tolerances because of the highly fluctuating levels of salinity in these habitats. Nonetheless, 25 %
salinity appears to be optimum for Thraustochytrid cultivation. Variations in salinity
significantly alter FAME profiles, with higher salinity favouring synthesis of LC-PUFAs. Along
with the sea water strength, temperature is another factor which needs to be adjusted for
optimum DCW and lipid production. Thraustochytrids grow well between 20-30°C with 25°C
considered the optimal temperature for cultivation. Increases in culture temperature significantly
alter FAME profiles, with increased production of LC-SFAs or LC-MUFAs, while a reduction in
temperature is reportedly associated with increases in DHA content but poor DHA productivity,
since growth is limited at low temperatures. When temperature was increased from 20 to 25°C, a
significant rise in palmitic acid and DCW productivity was observed but further increase in
temperature up to 30°C resulted in a reduction of DCW and palmitic acid, and DHA production
(Leaño et al. 2003).
38
2.5.6.7. FAME analysis of oil for biodiesel synthesis
Most of the lipids in Thraustochytrids and other oleaginous microorganisms are stored as TAGs
in lipid bodies (Scott et al. 2011). Thraustochytrids are reported to have more than 50 % oil as
SFA and MUFA, which are ideal for biodiesel production. Palmitic acid and DHA are the two
key fatty acids present in the FAME profile of reported Thraustochytrids. These two fatty acids
comprise as much as 70 %-90 % of total fatty acids. Myristic acid, stearic acid and oleic acid are
other fatty acids in SFA and MUFA. Linoleic acid, EPA and DPA are other fatty acids present in
the FAME profile. Oil is extracted and converted into fatty acid methyl/ethyl esters upon trans-
esterification facilitated by a catalyst. There are three types of catalyst system being primarily
used for trans-esterification reactions. These are alkaline, acidic, and acidic/alkaline.
NaOH/methanol, KOH/methanol, and NaOCH3 are the most frequently used catalyst systems
when free fatty acid (FFA) content is lower than 1mg.g-1 in the extracted lipid. In this method,
methanol and oils are mixed at a molar ratio of 6:1 for 1 h in the presence of NaOH at 60°C-
90°C catalysing the synthesis of FAMEs (Azócar et al. 2010). High content of FFAs upon
reaction with alkaline catalyst synthesizes soap and glycerol, which hinders the extraction of
FAMEs. In the case of higher FFA content, acid catalyst systems such as HCl/methanol,
H2SO4/methanol, and HCl/CH3COCl can be used for FAME production. Acid catalyst systems
are frequently used for trans-esterification of Thraustochytrids oil. Conversion inefficiency, the
need for high volumes of methanol and corrosion remains the foremost hurdles in this system
(Subramaniam et al. 2010). Heterogeneous systems have been developed to address these issues.
In these systems, acid catalysts are used in a pre-treatment step to convert FFAs into FAMEs
without forming soap, followed by alkaline reaction for final trans-esterification (Canakci et al.
2008).
39
Fig 2.4 shows an optimised FAME profile for biodiesel production, since fatty acid composition
significantly influences biodiesel properties such as viscosity, cetane number, density, cold flow
properties, oxidative stability and flash point (Miao et al. 2006). These properties can be
determined by carbon chain length, degree of unsaturation and quantity of each fatty ester
component in both fatty acids and triglycerides. Long carbon chain length and branched alcohol
moiety enhance viscosity, pour and cloud points of the fuel whereas higher degrees of
unsaturation appear to affect oxidative stability as well as the cetane number of the fuel
(Mutanda et al. 2011). According to European biodiesel standard EN 14214, the concentration of
acids containing four double bonds in FAMEs should be lower than 1 % for better oxidative
stability. Various reports suggest that fuel properties of microbial biodiesel are similar to those of
1st and 2nd generation energy crops and compatible with ASTM specifications for biodiesel
(Table 2.4).
Table 2.4. Comparison of different properties of microbial biodiesel and ASTM specification
(Johnson et al. 2009; Meher et al. 2006)
Properties Biodiesel (ASTM D6751)
Microbial biodiesel Diesel
Viscosity (mm2/s) at 40°C 1.9-6.0 2.5-5.2 1.9-4.1 Cloud point (°C ) -3 to 12 -11 -15 to 5 Pour point (°C ) -15 to 10 -15 -35 to -15 Flash point (°C ) 100 - 170 115 60 - 80
Lower heating value (MJ kg-1) 35 - 50 41 40 - 45 Cetane No. 48-65 - 40-55
Density (kgL-1) 0.880 0.864 0.85 Sulfur content (% w/w) 0-0.0024 0 <0.05
Acid value (mg KOH g-1) <0.5 0.374 <0.5 H/C ratio 2.02 1.81 1.81
40
Figure 2.4. Optimised FAME profile for biodiesel production
2.5.6.8. Manipulation of the FAME profile
Most of the fatty acids are distributed in TAGs, with a small fraction stored in the form of
membrane lipids such phospholipids and glycolipids. The quantity of non-TAGs is almost
insignificant in terms of biodiesel synthesis. Palmitic acid or DHA/DPA are the two major fatty
acids present in the lipid profile of Thraustochytrids and other ω-3 fatty acid producing
oleaginous microorganisms (Fan et al. 2009; Kobayashi et al. 2011). The presence of LC-
PUFAs, particularly DHA, DPA, and EPA, in the biodiesel will reduce oxidative stability, thus
requiring their removal from the oil. This can be achieved by either blocking the biosynthesis of
41
LC-PUFAs in microorganisms or by fractioning out LC-PUFAs from the oil. Delta-5 and Delta-6
fatty acid desaturases are the two key enzymes in PUFA synthesis, catalysing the conversion of
ALA/LA into EPA/AA. These enzymes can be targeted for the production of tailored fatty acids.
Approaches such as mutation, genetic engineering and enzyme inhibitors coupled with
optimisation of heterotrophic cultivation can be used to decrease the level of unwanted PUFAs in
the oil (Certik et al. 1999).
Application of desaturase inhibitors could be a handy tool for manipulation of the FAME profile.
Antioxidants such as propyl gallate, octyl gallate and curcumin [1,7-bis(Chydroxy-3 methoxy
phenyl)-1,6-heptadiene-3,5-dione)] are well-known inhibitors of Delta-5 and Delta-6 fatty acid
desaturases. These are non-competitive inhibitors with a significantly lower Ki value.
Kawashima and co-workers (1992, 1996) demonstrated a significant drop in LC-PUFAs and an
increase in the FAME profile of SFA and MUFA when rat liver microsomal cells and
Mortierella alpina were grown in medium containing alkyl gallates or curcumin (Kawashima et
al. 1996; Shimizu et al. 1992). These results are in agreement with the findings of Goldberg et
al. (1999), who observed significant changes in growth and lipid composition when
salicylhydroxamic acid (a delta-6 fatty acid desaturases inhibitor in microalgae) was used in a
culture of Porphyridium cruentum (Khozin-Goldberg et al. 1999). However, contrasting results
were obtained by Swarf et al. (2003), who documented no change in FMAE profile. In fact lipid
accumulation decreased when the Crypthecodinium cells were cultivated in the presence of alkyl
gallate (De Swaaf et al. 2003a). These reports suggest the existence of different regulatory
mechanisms for lipid biosynthesis in oleaginous microalgae such as Crypthecodinium or
Porphyridium. Therefore the mechanism of these inhibitors requires further study to assess the
effects on growth and fatty acid profile of the oleaginous microorganisms.
42
2.6. Thraustochytrids for carotenoid production
Carotenoids are an important group of natural pigments represented by over 600 novel biological
compounds (Scaife et al. 2009). Some of these pigments are well characterized, such as ß-
carotene and xanthophylls, due to their economic importance. These pigments used as natural
food colorants, nutraceuticals and feed supplements in the aquaculture and poultry industries.
Carotenoids are well known for their antioxidant properties, protecting against free radical
induced carcinogenesis, cardiovascular diseases and age related macular degeneration (Johnson
2002). Intake of a carotenoid supplemented diet could help reduce the risk of these disorders
(Bhosale 2004). Carotenoids are being chemically to meet the growing demand however
growing awareness about potential toxicity associated with synthetic carotenoids is leading an
increase in demand for natural carotenoids. Thraustochytrids are well documented for their
carotenoid production. Different carotenoids, such as ß-carotene and xanthophylls, have been
isolated from Thraustochytrids (Aki et al. 2003; Burja et al. 2006). Carotenoid content in these
microorganisms is overwhelmingly affected by type of carbon source and its quantity in the
cultivation medium. A Schizochytrium strain isolated by Aki and co-workers (2003) accumulated
up to 10 mgL-1 canthaxanthin in 4 days in a medium supplemented with 10 % glucose and 6 %
nitrogen at 50 % salinity, while astaxanthin production of 4.1 mgL-1 was maximum at low
nitrogen concentration (Armenta et al. 2006). The same strain when grown with 2 % glucose
produced 8 mgL-1 ß-carotene. Apart from altering culture conditions, mutation is another
important technique being used to enhance carotenoid production in Thraustochytrid strains.
Chatdumrong et al. reported an almost two fold increase in astaxanthin content when
Thraustochytrids cells were treated with N-methyl-N′-nitro-N-nitrosoguanidine NTG
(Chatdumrong et al. 2007). Although light is not necessary for Thraustochytrids cultivation,
43
carotenoids synthesis is impacted by different wavelength lights used in the growth of these
organism. A substantial drop in astaxanthin content was observed from cultures grown in the
presence of red to violet lights, while an opposite trend was observed for ß-carotene content
(Oclarit et al. 2009). Astaxanthin content was highest at the end of growth phase, reinforcing the
notion that this secondary metabolite is synthesized by the oxidation of ß-carotene under stress
conditions (Quilodrán et al. 2010).
44
CHAPTER.3
3. Isolation, characterization and heterotrophic cultivation study of a new
oleaginous marine yeast DBTIOC-ML3
3.1. Introduction
Biodiesel from oleaginous microorganisms has undergone considerable study since it is being
produced from biodegradable, non-toxic and renewable feedstocks and so has considerable
commercial potential. Biodiesel is compatible with existing transport infrastructure and can
substitute for petroleum derived liquid transport fuel (Sharma et al. 2011). Commercial success
of biodiesel from oleaginous microorganisms will depend on the long term supply of nutrients,
particularly carbon source, and the availability of large quantities of this source at a low cost.
Carbon sources including glucose, glycerol and volatile fatty acids (VFAs) are being used in
biodiesel production, but have not proven to fully satisfy the necessary conditions for
sustainability of the process.
Lignocellulosic materials are available in large quantity and are of interest as low cost carbon
sources for biofuel production. About 910 million metric tonnes of wheat straw was produced
worldwide in 2011, calculated by multiplying the global wheat production (approximate 701
million metric tonnes, FAO 2011) by a factor of 1.3 (Yu et al. 2011). Cellulose and
hemicellulose are the two major components of wheat straw, ranging from 35 %-45 % and 20 %-
30 %, respectively, followed by lignin (8-15%) (Yu et al. 2011). However, composition varies
by location. Glucose is predominantly present in cellulose and xylose in hemicellulose. These
sugars are tightly packed in the lignocellulosic matrix, reducing their accessibility to hydrolytic
enzyme and the release of sugar. Thus prior to enzymatic hydrolysis, pre-treatment is necessary
45
to increase the substrate accessibility and subsequent sugar release from the tightly packed
lignocellulosic matrix.
Hexose sugars are widely used as a carbon source for ethanol heterotrophic cultivation, applying
different strains of Sacchamyces cerevisiae (Jørgensen 2009), Kluyveromyces marxianus
(Talebnia et al. 2010) or Zymomonas mobilis (Lin et al. 2006) and process for hexose to ethanol
production is well established. Pentose sugars are the second most abundant sugar present in any
lignocellulosic material. However efficiency of pentose sugar fermentation is quite low, as
compare to hexose sugar fermentation, due to lack of enzymatic machinery necessary for pentose
fermentation in most of the natural strains of S. cerevisiae (Karakashev et al. 2007; Lin et al.
2006). S. cerevisiae strains were genetically modified to overcome this drawback. However
those strains showed little improvement in terms of ethanol yield, productivity and tolerance
(Karhumaa et al. 2007). Pentose sugars can be assimilated into lipid by oleaginous yeasts such as
Rhodotorula (Li et al. 2008), Lipomyces (Tapia V et al. 2012) and Trichosporon (Hu et al.
2011). Pentose assimilation to lipid by oleaginous yeast is efficient as compare to pentose
fermentation to ethanol by ethanologenic yeast. Energy density of lipid is higher when compared
to ethanol, thus it is more efficient to convert pentose sugars into lipid rather than ethanol.
Hemicellulose is the second most abundant polysaccharide (consisting of pentose sugars) after
cellulose in lignocellulosic materials. The conversion of pentose sugars into lipid together with
hexose fermentation to ethanol may provide an economical route for biofuel production.
Efficient conversion of lignocellulosic DCW into biofuel requires effective pre-treatment before
enzymatic hydrolysis. However, this pre-treatment can result in the formation of inhibitors such
as acetic acid, furfural and hydroxymethyl furfural (HMF). These inhibitors have adverse effects
on both ethanol heterotrophic cultivation (Amin et al. 1984; Palmqvist et al. 1999) and lipid
46
assimilation (Yu et al. 2011; Zhao et al. 2012). Liquid hydrolysates need to be detoxified to
remove inhibitors before heterotrophic cultivation or lipid assimilation. Detoxification processes
such as over liming, activated charcoal, neutralization or evaporation have been demonstrated to
be effective for inhibitor removal, but in the process also remove some of the sugars thereby
reducing yield. The detoxification step also enhances the energy footprint of the process,
resulting in higher input costs. Therefore it would be beneficial to isolate oleaginous yeast which
can convert pentose sugars into lipid in the presence of inhibitors.
In this chapter, we described the isolation and characterization of a fast growing strain of
oleaginous yeast Rhodotorula sp. DBTIOC-ML3 from Pichavaram mangroves, Tamilnadu. This
strain is capable of producing substantial amount of DCW and lipid utilizing non-detoxified
liquid hydrolysate (NDLH), without the need to remove inhibitors. Various strategies were
applied to enhance the productivity of this strain, using a variety of C/N ratios with either
glucose or xylose as the carbon source.
47
3.2. Materials and methods
3.2.1. Reagents and chemicals
All the chemicals and regents used in this study were either analytical or molecular grade.
Medium constituents including D-glucose, glycerol, acetic acid, xylose, yeast extract, peptone,
sodium nitrate, potassium nitrate, ammonium chloride, ammonium acetate and ammonium
sulphates were procured from Sigma-Aldrich, (St. Louis, MO, USA) or Himedia (Mumbai, MH,
India). Sea salt from Instant Ocean (Blacksburg, VA, USA) was added in the medium to mimic
the marine environment. Antibiotics including Penicillin G, Streptomycin, Rifampicin and
Nystatin were sourced from Sigma-Aldrich (St. Louis, MO, USA). For enzymatic profiling of
the strain an APIZYM kit was procured from Biomerieux India, (Delhi, India). Methanol,
chloroform, hexane, toluene from Fischer Scientific (Waltham, MA, USA), Methyl
nonadecanoate, Butylated hydroxytoluene, Potassium bicarbonate from Sigma-Aldrich (St.
Louis, MO, USA) were used in lipid extraction, FAME preparation and FAME analysis.
3.2.2. Sample collection and isolation of strain DBTIOC-ML3
Soil, water and degraded vegetation were collected in 50 mL sterile falcon tubes from
mangroves, estuary and open sea (Bay of Bengal) in the Pichavaram mangrove area of
Tamilnadu, India in June 2011. Physical parameters such as pH and temperature of the collection
site were recorded with pH/Temperature meter (pH56, Milwaukee, Rocky Mount, NC, USA).
100 μL of antibiotic mixture containing PenicillinG/Streptomycin (50 mg mL-1), Rifampicin (50
mg mL-1) and Nystatin (10 mg mL-1) was added into each falcon tube and samples were stored in
dry ice packs and brought to the lab together with natural sea water within 24 h for processing.
Soil samples were diluted 1000 times and leaf samples were washed with sterile sea water before
48
spreading onto agar plates. Water samples were directly spread onto agar plates. These agar
plates were supplemented with antibiotic mixture as described above. 100 μL of water was
carefully taken from the surface of the falcon tubes and spread on antibiotic containing agar
plates. Medium for these plates were contained glucose (10 gL-1), Yeast extract (1 gL-1),
mycological peptone (1 gL-1), agar (10 gL-1) in filtered natural sea water and antibiotic mixture
as described above. These plates were incubated at 25°C for 7-10 days. Axenic culture of the
strains was obtained by single colony picking and streaking onto agar plates pre-treated with
antibiotic mixtures.
3.2.3. Effect of different carbon and nitrogen sources on DCW, lipid production and fatty
acid profile for strain DBTIOC-ML3
Culture was maintained at 25°C on agar plate having glucose 5 gL-1, yeast extract 2 gL-1, peptone
2 gL-1, sea salt 18 gL-1 (to make 50 % sea water strength) and agar 10 gL-1. Plates were sub
cultured each 25 days. One μL of culture was taken from the plate and inoculated in seed
medium containing glucose 30 gL-1, yeast extract 10 gL-1, peptone 1gL-1, sea salt 18 gL-1. All the
medium used in this study were sterilized by autoclaving at 121°C for 15 min. Five percent v/v
of 48 h old inoculum was transferred into the production medium having a composition similar
to inoculum medium, except the carbon source, which was either glucose, glycerol, xylose or
acetic acid. In the next experiment, production medium was similar to inoculum medium, except
nitrogen sources, which were either yeast extract (YE), ammonium sulphates (AS), ammonium
acetate (AA), ammonium chloride (AC), sodium nitrate (SN), potassium nitrate (PN) or urea.
Cultures were incubated at 25°C, 4 day at 150 rpm for DCW production. The 4 day old cultures
49
were harvested and washed twice with distilled water before oven drying of the DCW at 90°C
for 24 h to 48 h, followed by gravimetric qualification of the dried DCW.
3.2.4. Impact of different C/N ratios on DCW, lipid production and fatty acid profile for
strain DBTIOC-ML3
Concentrations of the carbon source were varied from 30 gL-1, to 60 gL-1, to 90 gL-1, to 120 gL-1
and to 150 gL-1, along with 10 gL-1 yeast extract in production medium, to give C/N ratios of 13,
26, 39, 52 and 65. Glucose or xylose was used as the carbon source in the medium and yeast
extract as nitrogen source. The medium was sterilized by autoclaving at 106°C for 25 min. The
remaining medium composition and culture conditions were similar to those described in section
3.2.3. DCW was harvested after 4 days for lipid and fatty acid analysis in all the experiments,
unless otherwise described.
C/N ratio = (Wc * Mn)/(Wn * Mc)
Wc & Wn - Weight of carbon and nitrogen in the amount of carbon and nitrogen source
respectively, Mc & Mn - Molecular weight of carbon and nitrogen atom respectively
3.2.5. DCW, lipid production and FAME profile for growth of strain DBTIOC-ML3 on
non-detoxified liquid hydrolysate of wheat straw or synthetic hydrolysate (SH)
Synthetic hydrolysate was prepared by mixing the proportions of sugars as present in NDLH.
Synthetic wheat hydrolysate, or neutralized NDLH, was supplemented with 10 gL-1 yeast extract,
1 gL-1 peptone and 18 gL-1 sea salt, and sterilized by autoclaving at 121°C for 15 min. Sterilized
medium was further centrifuged at 12000 rpm for 30 min to remove precipitates formed during
autoclaving, and 50 mL medium was transferred aseptically to a 250 mL flask. Five per cent v/v
50
of 48 h old culture was inoculated in the production medium and incubated at 25°C for 4 day at
150 rpm for DCW production. DCW was harvested after 4 days and dried at 90°C for 24 h to 48
h followed by gravimetric quantification
3.2.6. Fermenter studies of strain DBTIOC-ML3 in a 2L continuous stirred tank reactor
Production medium (1 L) containing neutralized NDLH was inoculated with 5% v/v of 48 h old
yeast culture for DCW and lipid production in a 2L bioreactor (Bioflow/Cellgen 115, Eppendorf,
Hamburg, Germany). All the culture parameters were the same as described in section 3.2.2.,
except dissolved oxygen, agitation and pH which were maintained at 50 %, 250 rpm and 7,
respectively. Dissolved oxygen was adjusted to 50 % with the help of air and pure oxygen using
inbuilt mass flow controllers of bioreactor, whereas agitation remained fixed throughout the
culture. For growth profile, 25 mL of sample was harvested at 0 h, 10 h, 24 h and 40 h, for
DCW, lipid, residual sugar and inhibitor estimation. In a CSTR, the first harvesting (950 mL
culture) was done after 40 h, followed by addition of fresh medium (950 mL) in 2L vessel.
Subsequent harvestings were done at the interval of 24 h. 950 mL of culture was harvested at the
end of each cycle. DCW, lipid content and fatty acid composition were determined at 24 h
interval. The experiment was carried out twice and values are given as mean±SE.
3.2.7. Enzymatic profiling of the strain DBTIOC-ML3
Five per cent of 48 h old culture was inoculated in production medium containing glucose (1 gL-
1), yeast extract (0.1 gL-1), peptone (0.1 gL-1), tryptone (0.1 gL-1) and sea salt (18 gL-1 to simulate
50 % v/v sea water strength). Five day old culture was harvested by centrifugation at 4000 rpm
for 15 min and washed twice with sterile artificial sea water to remove traces of medium
51
components. Pellet was dissolved in sterile artificial sea water and cell density was adjusted
between 4-10× 105 cells mL-1 with sea water. 65 μL of culture was added into each well of the
strip and incubated at 20°C for 15 h-16 h. 30 μL of solution Zym A (provided with the API ZYM
kit) was added into the well and incubated at 20°C for 5 min, followed by addition of Zym B (30
μL) and incubation at 20°C for 15 min. Colour started to develop after the addition of solution
Zym B. Colour intensity was recorded on a scale of 1-5, according to the reading colour scale
provided with the kit for semi-quantification of enzyme activities.
3.2.8. Molecular identification of strain DBTIOC-ML3
Genomic DNA was extracted from 500 μL of 3 day old culture, according to the protocols
described in the DNeasy blood and tissue kit (Qiagen, USA). Purified genomic DNA was used as
template for PCR amplification of 18S rDNA with primers T18S1F 5′-
CAACCTGGTTGATCCTGCCAGTA-3′ and T18S5R 5′-
TCACTACGGAAACCTTGTTACGAC-3′ (Honda et al. 1999). 25 μL PCR reaction, comprising
of 12.5 μL PCR master mix (TaqMan, Applied Biosystem), 0.5 μL each primer (T18S1F,
T18S5R), 1 μL genomic DNA and 10.5 μL milliQ water, was set up. The PCR program included
initial denaturation for 3 min at 94°C, final denaturation for 45s at 94°C, annealing for 30s at
64°C, 30 cycles, 2 min at 72°C for extension and final extension for 10 min at 72°C. The PCR
product was purified on 1 % agarose gel using a QiAquick gel extraction kit (Qiagen, USA). The
sequencing reaction mix of PCR product, primers and milliQ water was sent to Macrogen (South
Korea) for 18S rRNA gene sequencing. The resulting sequence was analysed with known
Rhodotorula 18S rRNA gene sequences obtained from the NCBI gene bank database, using
52
BLAST. A phylogenetic tree (NJ tree) for the strain was constructed using MEGA 5.1 software,
with other known sequences of Rhodotorula obtained from the NCBI database.
3.2.9. Sugar and inhibitor analysis of wheat straw NDLH with HPLC
Liquid hydrolysate of pre-treated wheat straw was neutralized according to the protocol
described by (Yu et al. 2011). Calcium hydroxide was added into the NDLH until the pH
reached 7 while stirring at 30°C, followed by centrifuge at 12000 rpm for 30 min to remove
precipitate. One mL of NDLH was filtered with a syringe filter and injected into the HPLC
system (Waters, USA) equipped with refractive index detector and Biorad Aminex HPX-87H
column (Bio-Rad, USA) for peak identification and quantification. Sulphuric acid (0.005 M) was
used as the Mobile phase with a flow rate of 0.6 mL min-1. Peaks were identified and quantified
based on retention times and peak area of standard of carbon sources and organic acids. A
calibration curve was prepared for glucose, glycerol, xylose, acetate acid, malic acid, lactic acid
by plotting different concentrations (100 ppm, 500 ppm, 1000 ppm and 10000 ppm) on x-axis
and their respective peak areas on y-axis.
3.2.10. Lipid extraction and FAME analysis
For lipid extraction, the protocol as reported by Gupta et. al. was followed with some
modifications (Gupta et al. 2012b). Dried biomass (10 mg) was suspended in 600 μL solvent
(chloroform: methanol, 2:1) in a 2 mL centrifuge tube. Chloroform was added first followed by
methanol and this was vortexed for 3 min before centrifuging at 13000 rpm for 15 min. This
process was repeated 2-3 times for complete extraction. Hexane was added into the lipid extract
and the upper layer was harvested carefully with a Pasteur pipette, followed by evaporation
53
under nitrogen. Lipid was measured gravimetrically. For FAME analysis, 500 μL of toluene was
added into the dried lipid extract followed by addition of a 100 μL internal standard (methyl
tricosanoate C19:0) and 100 μL butylated hydroxytoluene (BHT). Acidic methanol (400 μL) was
added into the tube and kept for 10-12 h at 50 °C. One mL of 5 % NaCl was added followed by
the addition of 1 mL of hexane and the upper layer was transferred into a fresh tube. FAMEs
were washed with 1 mL of 2 % potassium bicarbonate and moisture was removed by passing this
through anhydrous sodium sulphate. FAMEs were concentrated under nitrogen and analysed
with a GC-FID system (Perkin Elmer clarus680, US), equipped with a fast-GC capillary column
(Ωwax100, 15 m x 0.1 mm, 0.1 μm thickness). One μL of FAME was injected at an injector
temperature 250 °C. Oven temperature was increased from 140°C to 280 °C with a ramping rate
of 40 °C/sec and held for 2 min. Detector was set at 260 °C. Hydrogen was used as a carrier gas
with a velocity of 50 cm sec-1. In Totalchrome chromatography software (Perkin Elmer, US),
fatty acid peaks were identified with the help of elution time of standard fatty acids and
quantified based on peak areas in relation to internal standard.
54
3.3. Results and discussion
3.3.1. Selection and identification of strain DBTIOC-ML3
From Pichavaram samples, a total of 25 strains were purified based on morphology visualized
under a phase contrast microscopy. These strains were rigorously screened for DCW and lipid
production. One strain was selected for further study from 25 strains, since this strain DBTIOC-
ML3 was observed to produce the highest DCW and lipid content. Phase contrast imaging of this
strain revealed a cell size of between 5 μm to 8 μm, with an ellipsoidal cell shape, when
visualized under 40X magnification. The strain produced a dark orange colour on the agar plate
and in broth, similar to that reported by Gupta et al (Gupta et al. 2012b), together with a similar
fatty acid profile.
Figure 3.1. Phylogenetic tree of strain Rhodotorula sp. DBTIOC-ML3
To confirm the identity of the strain, 18S rDNA was amplified and sequenced followed by
BLAST analysis. The resulting sequence was deposited in the NCBI database with sequence id
55
KJ528560. BLAST analysis of 18S rDNA of this strain revealed it has 99 % similarity with
Rhodotorula mucilaginosa (KC674229.1). A neighbour joining tree (NJ tree) was prepared for
this strain using existing 18S rDNA sequences from the NCBI database to establish its
phylogenetic relationship. Phylogenetic analysis (NJ tree) of this strain with existing 18S rDNA
sequences in the NCBI database revealed its close proximity with an existing Rhodotorula sp.
(Fig 3.1).
3.3.2. Enzymatic profiling of the strain DBTIOC-ML3
API-ZYM identification kits are a rapid method for screening strains producing industrially
significant enzymes such as lipases and β-glucosidase. The API-ZYM enzyme kit was used for
semi-quantification of enzymatic activities of DBITOC-ML3. In the enzymatic reaction test this
strain showed positive activity for 19 hydrolytic enzymes (Fig 3.2). The strain exhibited very low
activity (scale 1 on reading colour scale) for esterase, lipase, trypsin, α-chymotrypsin, α-
galactosidase, β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase and
α-fucosidase. Higher activity was observed for the enzymes naphthol-AS-BI-phosphohydrolase,
alkaline phosphatases and acid phosphatases (scale 4-5 on reading colour scale). Other enzymes
including α-glucosidase, β-glucosidase leucine arylamidase and valine arylamidase were also
detected but their activity was in the mid-range (scale 2-3 reading colour scale).
56
Figure 3.2. Enzymatic profiling of cell bound enzymes of strain DBTIOC-ML3
3.3.3. DCW and lipid production for strain DBTIOC-ML3 using a variety of carbon
sources
Different carbon sources such as glucose, glycerol, xylose, acetic acid were used in this work to
assess the ability of the newly strain yeast strain to utilize them to produce DCW and lipid.
57
Glucose is normally the preferred carbon source for microbial growth. Addition of glucose in the
medium resulted in relatively high DCW levels of 18.42 gL-1 with 22 % lipid (Table 3.1), which
was significantly higher than previous reports for Rhodotorula glutinis by (Braunwald et al.
2013). They reported production levels of 11.4 gL-1 DCW and 10% -15 % lipids when using 6%
glucose, which was in agreement with findings of Zhan et. al. on Rhodotorula glutinis using 3%
sugar (Zhang et al. 2011), where they found maximum production of 6.25 gL-1 DCW and 16%
lipid. Productivity of strains vary from strain to strain and the higher DCW productivity of our
strain is not surprising. However, the use of pure glucose for lipid production is not economically
viable for biofuel production and so low cost glycerol is preferable. With increased production of
biodiesel, increased levels of crude glycerol are generated (Ethier et al. 2011). Crude glycerol
contains a range of impurities such as soap and methanol and so it is costly to purify this glycerol
for use in cosmetics, food or pharmaceuticals. Disposal of such large amount of glycerol is an
important issue that needs to be dealt with. Using crude glycerol as a carbon source for DCW
and lipid production is one of the proposed methods for recycling glycerol (Chi et al. 2007b).
Use of glycerol as a carbon source resulted in comparable DCW (19.85 gL-1) to that produced
with glucose (18.42 gL-1), but lipid content decreased from 22 % in glucose to 18.5 % glycerol
(Table 3.1).
A similar pattern was observed by Galafassi et al (2012) when they were studying the effect of
different carbon sources on productivity of Rhodotorula graminis (Galafassi et al. 2012). DCW
production in this work is greater than previous reports on Rhodotorula sp. using glycerol as the
carbon source (Galafassi et al. 2012; Saenge et al. 2011; Yen et al. 2012).
Xylose and acetic acid are generated in large quantity during pre-treatment of lignocellulose
DCW to produce the hemicellulose fraction, leaving behind cellulose rich DCW (Talebnia et al.
58
2010). Conversion of xylose into ethanol is inefficient as compare to hexose fermentation,
whereas acetic acid inhibits ethanol fermentation (Lin et al. 2006). However these two carbon
sources can be efficiently converted into lipid with oleaginous yeast (Yu et al. 2011). Literature
suggests a significant drop in DCW and lipid production occurs when xylose is used as a carbon
source in place of glucose or glycerol (Galafassi et al. 2012; Hu et al. 2011; Zhang et al.
2011).When glucose or glycerol was replaced with xylose in medium, almost equal DCW was
obtained. However in our strain, lipid content using xylose was almost 20 % higher than that
produced using glucose and 40 % higher than that produced using glycerol (Table 3.1).
Table 3.1. DCW and lipid production using different carbon sources (n=2, mean±SE)
Carbon source (3%) DCW (gL-1)
Lipid (% of DCW)
Lipid (gL-
1) DCW yield
(Xb)3
Lipid yield (XL)4
DCW productivity
(Yb)5
Lipid productivity
(YL)6
Glucose 18.42±0.27 22.0±1.0 4.05 0.61 0.13 4.60 1.01
Glycerol 19.85±0.60 18.5±1.5 3.67 0.66 0.12 4.962 0.92
Xylose 17.51±0.35 26.5±1.5 4.64 0.58 0.15 4.37 1.16
Acetate 11.19±0.13 21.0±0.1 2.35 0.37 0.08 2.80 0.59
SH 10.41±0.57 18.0±1.0 1.87 0.41 0.07 2.60 0.47
NDLH 10.58±0.06 20.5±0.5 2.16 0.42 0.08 2.64 0.54
3 Xb – DCW yield (gram DCW produced from per gram of carbon source)
4 XL – Lipid yield (gram lipid produced from per gram of carbon source)
5 Yb – DCW productivity (gram DCW produced per litre per day)
6 YL - Lipid productivity (gram lipid produced per litre per day)
59
These results indicate that our strain can assimilate xylose more efficiently than other
Rhodotorula strains reported in the literature. Addition of acetate as the sole carbon source in the
medium resulted in a significant drop in DCW (11.19 gL-1), although lipid content (21%) was
similar to that produced using glucose. This particularly significant since acetate is an
inexpensive substrate generated from numerous industrials applications, not only from the pre-
treatment of lignocellulosic DCW (Perez-Garcia et al. 2011). The ability of this strain to produce
higher DCW and lipid while assimilating acetate or xylose, at similar levels to glucose, makes
this strain applicable for DCW and lipid production from hydrolysates of pre-treated
lignocellulosic biomass, which contains predominantly glucose and xylose with some acetate.
3.3.4. Effect of different nitrogen sources on DCW and lipid production
Nitrogen source type and content in the medium influence the DCW and lipid productivity
during heterotrophic cultivation since nitrogen content determines growth rate in the initial phase
of the culture, followed by a lipid accumulation phase triggered by nitrogen exhaustion in the
medium. Different nitrogen sources are taken and metabolized through separate pathway (Zheng
et al. 2012). In this work, different organic and inorganic nitrogen sources, including yeast
extract, ammonium sulfate, ammonium chloride, ammonium acetate, potassium nitrate, sodium
nitrate and urea were used as nitrogen source in the medium. Maximum DCW was observed with
yeast extract (18.38 gL-1) and the lowest levels observed with urea or nitrates (5.72 gL-1 or 5.68
gL-1, respectively) (Table 3.2). Addition of inorganic nitrogen sources into the medium resulted
in decreased DCW production. However lipid content was comparable in all the nitrogen
sources, with maximum lipid content at 28.5 % of DCW when using ammonium acetate. Evans
et. al. observed a similar trend while culturing 17 oleaginous yeast on different organic and
60
inorganic nitrogen sources (Evans et al. 1984). In addition to being a nitrogen source, yeast
extract has other nutrients such as vitamins and growth factors. The supply of these micro
nutrients may be the reason for higher DCW production when using yeast extract as the nitrogen
source (Ren et al. 2013a). Highest DCW and lipid yield (0.61 and 0.13 respectively) along with
productivity (4.59 gL-1d-1 DCW and 0.94 gL-1d-1 lipid) was achieved with yeast extract as the
nitrogen source. Use of ammonium sulfate as the nitrogen source resulted in the second highest
yield (0.40 for DCW and 0.09 for lipid) and productivity (2.98 gL-1d-1 DCW and 0.67 gL-1d-1
lipid) after yeast extract. Initial pH of the medium was around 6.5-7.0 irrespective of the nitrogen
source. However the final pH of the supernatant varied dramatically, with the lowest pH
observed with ammonium sulfate (pH 2.02) and ammonium chloride (pH 1.86) and highest with
urea (pH 9.23). This strain exhibited strong pH tolerance and grew on a broad range of pH from
1.88 to 9.23, with the optimum pH at 6-7. Variation from this range caused decrease in DCW
production even though lipid content was comparable in all the cultures (Ren et al. 2013a). Final
pH of the residual medium provides information about the pattern of utilization of different
nitrogen sources in the medium, with maximum uptake of ammonium ions by cells leaving
behind strong anion which result in a significant drop in final pH, for example in medium
containing AS, AC, PN and SN (Table 3.2).
61
Table 3.2. DCW and lipid production on different nitrogen sources (n=2, mean±SE)
Nitrogen source (3%) DCW (gL-1)
Lipid (% of DCW)
Lipid (gL-1)
DCW yield (Xb)
Lipid yield (XL)
DCW productivity
(Yb)
Lipid productivity
(YL) YE 18.38±0.82 21.0±1.0 3.86 0.61 0.13 4.59 0.96
AS 11.93±0.57 22.5±0.5 2.68 0.40 0.09 2.98 0.67
AC 10.42±0.40 26.5±2.5 2.76 0.35 0.09 2.60 0.69
AA 8.14±0.70 28.5±0.5 2.32 0.27 0.08 2.03 0.58
PN 6.11±0.27 23.5±0.5 1.43 0.20 0.05 1.53 0.36
SN 5.68±0.26 23.5±1.5 1.33 0.19 0.05 1.42 0.33
Urea 5.72±0.44 16.0±22.0 0.91 0.19 0.03 1.43 0.23
3.3.5. Effect of increasing concentration of glucose or xylose on DCW and lipid production
Concentrations of glucose or xylose of 30 gL-1, 60 gL-1, 90 gL-1, 120 gL-1 or 150 gL-1 were added
to the medium to make C/N ratio 13, 26, 39, 52 or 65 respectively. Higher C/N ratios causes
nitrogen stress and ample supply of carbon source was required to trigger lipid accumulation in
the cell, resulting in higher DCW and lipid production (Ratledge 2004). Increase in the C/N ratio
from 13 to 26 using glucose as the carbon source translated into higher DCW and lipid
production from 18.36 gL-1 DCW and 24.5 % lipid with C/N ratio 13 to 23.62 gL-1 DCW and 40
% lipid with C/N ratio 26, which resulted in increased DCW and lipid productivity from 4.59 gL-
1d-1 and 1.12 gL-1d-1 with C/N ratio 13 to 5.50 gL-1d-1 and 2.36 gL-1d-1 with C/N ratio 26 (Table
3.3). Lipid productivity almost doubled with doubling C/N ratio. Further increase in the C/N
ratio from 13 to 65 did not translate into higher DCW or lipid content although residual glucose
62
in the medium increased significantly from 21.35 gL-1 under a C/N ratio 13 to 105.10 gL-1 under
a C/N ratio 65.
Similar growth patterns were observed when xylose was used as the carbon source in place of
glucose. Maximum DCW (27.85 gL-1), lipid content (34.0 %), DCW productivity (4.46 gL-1d-1)
and lipid productivity (2.36 gL-1d-1) was recorded with a C/N ratio 26. As the C/N ratio was
increased from 26 to 65, a decrease in growth DCW and lipid productivity was observed. Lowest
DCW and productivity (2.95 gL-1d-1 and 0.65 gL-1d-1) was recorded at a C/N ratio of 65 (Table
3.3). The reason behind lower DCW production, lipid content and DCW yield may be the
inability of the strain to utilize high concentration of sugar present in the medium. Decrease in
pH of the medium (up to 4) was observed with increasing C/N ratio, which is in agreement with
the findings of Ren and co-workers (Ren et al. 2013a). They reported decrease in DCW and lipid
when pH of the medium was changed from 7 to 4. Literature suggests that lowering the pH will
disrupt the hexose/H+ symport system which is responsible for the transport of hexoses into the
cell (Perez-Garcia et al. 2011). This could be the reason behind the inability of this strain to
utilize higher concentrations of glucose once the pH of the medium deviates from optimal pH.
63
Table 3.3. Effect of C/N ratio on DCW and lipid production (n=2, mean±SE)
Carbon source
C/N ratio
DCW (gL-1)
Lipid (% of DCW)
Lipid (gL-1)
DCW yield (Xb)
Lipid yield (Xl)
DCW productivity
(Yb)
Lipid Productivity
(Yl)
Residual
sugar (gL-1)
Glucose 13 18.36±0.66 24.5±2.5 4.50 0.61 0.15 4.59 1.12 0
26 23.62±0.45 40±2.0 9.45 0.61 0.24 5.90 2.36 21.35±0.35
39 21.15±0.69 24±1.0 5.07 0.58 0.14 5.29 1.26 53.8±0.40
52 17.73±1.27 24.5±0.5 4.34 0.54 0.13 4.43 1.08 87.55±4.35
65 14.94±0.51 23±3.0 3.44 0.33 0.08 3.73 0.86 105.1±9.70 Xylose 13 17.71±0.23 22.5±2.5 3.98 0.59 0.13 4.43 0.99 0
26 27.85±0.45 34±1.0 9.47 0.65 0.22 4.46 2.36 16.9±3.10
39 14.16±0.38 24.5±2.5 3.47 0.38 0.09 3.54 0.86 53.1±2.30
52 13.22±0.20 22.5±1.5 2.97 0.39 0.09 3.30 0.74 86.0±2.80 65 11.82±0.28 22±2.0 2.60 0.34 0.07 2.95 0.65 115.2±5.60
3.3.6. NDLH and SH as carbon sources for DCW and lipid production
The hemicellulose fraction of acid pre-treated lignocellulosic streams contains primarily xylose
and smaller amounts of glucose and arabinose (Yu et al. 2011). HPLC analysis of NDLH
revealed the significant presence of these sugars, in the order xylose (16.57 gL-1), glucose (5.58
gL-1) and arabinose (0.95 gL-1). NDLH also contained inhibitors such as acetic acid (2.35 gL-1),
HMF (0.62 gL-1) and Furfural (3.10 gL-1) (Table 3.4). Acetic acid can be used as a carbon source
for lipid production in some oleaginous microorganisms (De Swaaf et al. 2003b). SH of wheat
straw was prepared mimicking the composition of total fermentable sugars present in NDLH, to
explore the effect of acetic acid, HMF and furfural on DCW and lipid production and fatty acid
composition.
64
Table 3.4. Sugar and inhibitor concentration of NDLH of dilute acid pre-treated wheat straw, analysed by HPLC (n=2, mean±SE)
Sugar/Inhibitor Concentration (gL-1)
Glucose 5.58±0.24 Xylose 16.57±0.51
Arabinose 0.95±0.05 Acetic acid 2.35±0.15
HMF 0.62±0.01 Furfural 3.10±0.20
Addition of NDLH or SH into the medium as the carbon source resulted in almost equal DCW,
with 10.58 gL-1 DCW and 20.5 % lipid for NDLH and 10.41 gL-1 DCW and 18 % lipid for SH
(Table 3.1). HMF and furfural showed no growth inhibitory effect on DCW, but had stimulatory
effects on lipid production in our strain Rhodotorula sp. DBTIOC-ML3. Similar growth patterns
were observed in previous reports by (Yu et al. 2011) and (Galafassi et al. 2012). Galafassi et. al.
2012 also reported complete inhibition of growth of R. graminis when medium was
supplemented with 2.5 gL-1 furfural and/or 3gL-1 acetic acid. NDLH used by Yu et. al. 2011 for
R. glutinis cultivation contained low inhibitor concentrations (acetic acid 4 gL-1, HMF 0.05 gL-1,
furfural 0.44 gL-1), as compare to those used in this work (acetic acid 3.92 gL-1, HMF 0.62 gL-1,
furfural 3.10 gL-1). Comparing with literature data Rhodotorula sp. DBTIOC-ML3 has higher
inhibitor tolerance than previous reports. HPLC analysis of 4th day supernatant had also showed
complete utilization of acetic acid for growth rather than inhibiting growth. Therefore, use of this
strain should provide operational flexibility when using non detoxified hemicellulose fractions
for lipid production having low or higher inhibitor concentration.
65
3.3.7. Fatty acid composition of strain DBTIOC-ML3 under different culture conditions
Fatty acid analysis of strain DBTIOC-ML3 revealed palmitic acid (C16:0) and oleic acid (C18:1)
comprised 70 % to 90 % of total fatty acids (TFAs), irrespective of which carbon or nitrogen
sources were used for growth. Oleic acid constituted 40 % to55 % of TFA with palmitic acid at
30 % to40 % of TFA (Fig 3.3a). Pamitoleic acid (C16:1) and stearic acid (C18:0) are the other
major fatty acids present, with traces of linoleic acid and linolenic acid. Glucose, xylose and
acetate appear to favour the production of oleic acid (around 43 % to 50 % of TFA) over palmitic
acid (around 28 % to 41 % of TFA). However an opposite trend was observed when glycerol was
added into the medium as the carbon source, where the biosynthesis of palmitic acid increased
(46 % of TFA) as compared to oleic acid (28 % of TFA) (Fig 3.3a).
Addition of different nitrogen sources into the medium did not change the average composition
of oil, with palmitic acid and oleic acid remained the major fatty acids present. FAME profile of
lipids extracted from DCW grown on yeast extract and ammonium sulphate was similar, with the
majority of fatty acids being palmitic acid (almost 34 % to 41 % of TFA) and oleic acid (42 % to
45 % of TFA). Synthesis of palmitic acid and palmitoleic acid were similar with 24 % and 34 %
of TFA, respectively (Fig 3.3b). The maximum amount of palmitic acid was produced when
yeast extract was used as the nitrogen source, while the greatest amount of oleic acid was
achieved using ammonium chloride as the nitrogen source. Palmitic acid content was around 30
% of TFA, while oleic acid remained at around 50 % of TFA. Other nitrogen sources such as AS,
AC, AA, SN, PN and urea resulted in similar fatty acid profiles. Irrespective of carbon source, an
increase in the C/N ratio resulted in palmitic acid (C16:0) and oleic acid (C18:1) constituting
almost 90 % of TFA. Palmitic acid content remained nearly constant (38 %-42 % of TFA) while
oleic acid content increased from 37 % to 52 % of TFA as the C/N ratio increased from 3 to 15
66
(Fig 3.4a). Xylose appears to favour oleic acid production over palmitic acid. Oleic acid content
was higher in cultures fed with xylose, as compared with glucose, (37 % of TFA with glucose
and 46 % of TFA with xylose), irrespective of the C/N ratio (Fig 3.4b). Maximum oleic acid
content occurred with xylose having a C/N ratio 52. The high levels of palmitic acid and oleic
acid, along with the very low levels of PUFAs makes the lipids from this oleaginous yeast an
attractive feedstock for biodiesel production.
Figure 3.3. Fatty acid composition of strain DBTIOC-ML3 cultivated on (a) different carbon sources and (b) different nitrogen sources (n=2, mean±SE), C16:0 palmitic acid, C16:1 palmitoleic acid, C18:0 stearic acid, C18:1 Oleic acid, C18:2 Linoleic acid, C18:3 Linolenic acid
0
10
20
30
40
50
60
C16:0 C16:1 C18:0 C18:1 C18:2 C18:3
(a).
Fatty
aci
ds (%
of T
FA)
Fatty acids
Glucose
Xylose
Glycerol
Acetate
NDLH
Synthetic hydrolysate
0
10
20
30
40
50
60
C16:0 C16:1 C18:0 C18:1 C18:2 C18:3(b).
Fatty
aci
ds (%
of T
FA)
Fatty acids
Yeast extract
Ammonium sulphate
Ammonium chloride
Ammonium acetate
Potassium Nitrate
Sodium Nitrate
Urea
a
b
67
Figure 3.4. Fatty acid composition of strain DBTIOC-ML3 using medium containing different C/N ratio, with (a) glucose or (b) xylose as carbon source and yeast extract as nitrogen source (n=2, mean±SE), C16:0 palmitic acid, C16:1 palmitoleic acid, C18:0 stearic acid, C18:1 Oleic acid, C18:2 Linoleic acid, C18:3 Linolenic acid
3.3.8. Growth profile of strain DBTIOC-ML3 using NDLH as the carbon source
Growth profile of the strain showed very short lag phase at approximately 3 h to 4 h, reflecting
the ability of the strain to quickly acclimatize to the medium having significant amount of
furfural (3.10 gL-1) and HMF (0.62 gL-1). DCW increased exponentially after 5 h with rapid
depletion of sugars in the medium. In the first 10 h total sugars depleted from 25.45 gL-1 to 14.86
gL-1 with simultaneous utilization of glucose and xylose (Fig 3.5). This resulted in a sharp rise in
DCW to 5.48 gL-1 at 10 h. However, lipid content remained unchanged at 10 h reflecting the
0
10
20
30
40
50
60
70
C16:0 C16:1 C18:0 C18:1 C18:2 C18:3
Fatty
aci
ds (%
of T
FA)
Glucose C/N 13 C/N 26 C/N 39 C/N 52 C/N 65
01020304050607080
C16:0 C16:1 C18:0 C18:1 C18:2 C18:3
Fatty
aci
ds (%
of T
FA)
Fatty acids
Xylose C/N 13 C/N 26 C/N 39 C/N 52 C/N 65
68
nitrogen availability in the medium. Once the nitrogen source is depleted in the medium, lipid
accumulation is expected to rise (Gupta et al. 2013a; Singh et al. 2013), and this was observed
after 10 h. Lipid production increased from 0.85 gL-1 at 10 h to 4.09 gL-1 at 24 h with the increase
in DCW from 5.48 gL-1 at 10 h to 10.79 gL-1 at 24 h. At this time 80 % of the total sugar was
consumed with complete depletion of glucose and acetate in the medium. Surprisingly, the HMF
content almost doubled from 0.62 gL-1 at 0 h to 1.10 gL-1 at 24 h and further increased up to 1.39
gL-1 at 40 h together with complete consumption of furfural within 24 h. This result is in
agreement with the finding of Yu et al.2011(Yu et al. 2011), where they reported a sharp
increase in HMF content along with complete consumption of furfural within 24 h. Carbon
source was completely exhausted at 40 h so the culture was harvested, resulting in 14.41 gL-1
DCW and 6.06 gL-1 of lipid, with DCW productivity at 8.65 gL-1d-1 and lipid at 3.63 gL-1d-1.
Figure 3.5. Growth profile of strain DBTIOC-ML3 on NDLH in a 2L fermenter (n=2, mean±SE)
0
2
4
6
8
10
12
14
16
18
0
5
10
15
20
25
30
0 10 20 30 40
Lipi
d, D
CW
(gL-
1)
Sug
ar, I
nhib
itors
(gL-
1)
Time (h)
Glucose XyloseArabinose Acetic acidHMF FurfuralTotal sugar BiomassLipid
69
Encouraged from this finding, a semi-continuous heterotrophic cultivation was designed using
NDLH as the carbon source. In the first cycle, strain DBTIOC-ML3 took 40 h to completely
metabolize the carbon in the medium, producing 14.41 gL-1 DCW and 6.059 gL-1 lipid (Fig 3.6).
Figure 3.6. HPLC profile of fresh NDLH medium (inoculated with strain DBTIOC-ML3) and
depleted medium (40 h old) in a 2 L fermenter
However in next cycle, the strain took only 24 h to metabolize total carbon giving 14.90 gL-1
DCW and 6.15 gL-1 lipid. Once the strain was acclimatized in the new medium it consumed the
carbon source more rapidly. This may explain the higher DCW and lipid productivity observed
in the 2nd cycle. A similar trend was observed in the 3rd cycle with 13.90 gL-1 DCW and 5.89 gL-
1 lipid produced, confirming that strain DBTIOC-ML3 can utilize NDLH once it has adapted to
the medium (Table 3.5). Contrary to the inhibitory effect expected for HMF, furfural and acetate
70
during ethanol heterotrophic cultivation of S.Cerevisiae using NDLH as carbon source (Lin et al.
2006), we observed no negative effect of HMF on DCW and lipid production in our strain, and
furfural and acetate was totally consumed during heterotrophic cultivation.
Table 3.5. DCW and lipid production in semi-continuous culture (n=2, mean±SE)
Harvesting number
Time (h) Total DCW (gL-1)
Total lipid (gL-1)
DCW productivity (Yb)
Lipid productivity (YL)
1st 40th 14.41±1.91 6.06±0.45 8.65±1.12 3.03±0.26
2nd 64th 14.90±1.58 6.15±0.34 14.90±1.58 6.15±0.34
3rd 88th 13.90±1.98 5.89±0.65 13.90±1.98 5.89±0.65
71
3.4. Conclusion
This work demonstrated the ability of new strain of oleaginous yeast, Rhodotorula sp.DBTIOC-
ML3, to efficiently utilize hemicellulose fraction of acid pre-treated wheat hydrolysate, to
produce substantial amount DCW and lipid. The high tolerance of this strain to HMF, acetate
and furfural is unexpected and indicates that good lipids yields can be obtained without the need
for detoxification of the hydrolysate used as a source of carbon for growth. The absence of a
detoxification step did not compromise productivity during heterotrophic cultivation thus
elimination of detoxification step would reduce the cost associated with removal of inhibitors
from hydrolysate. This work showed the potential to integrate this process with ethanol
fermentation, where hemicellulose stream (rich in xylose sugar) coming out of pre-treatment of
hydrolysate can be fed into bioreactor for lipid production by oleaginous yeast, while hexose
sugar (glucose predominantly) can be directed for the ethanol fermentation by S.cerevisiae.
72
CHAPTER.4
4. Isolation and screening of Thraustochytrids from Indian and Australian
biodiversity for ω-3 fatty acids and biodiesel production
4.1. Introduction
Microbial oils are being prioritized due to increasing fossil fuel prices and their unsustainable
long term supply and security (Chisti 2013). Additionally, some of these microbial oils have
nutritional value since they are free from toxic impurities that may be present in fish oil, the
current source of high value polyunsaturated fatty acids (PUFAs) (Hauvermale et al. 2006).
Thraustochytrids are marine heterotrophic, fast growing protists and are well documented for
their high DCW and lipid producing ability. Thraustochytrids can produce a high content of ω-3
PUFAs in their oil, particularly Docosahexaenoic acid (DHA C22:6n3), although some strains
also produce Eicosapentaenoic acid (EPA C20:5n3). DHA and EPA have application as infant
formula, functional food, nutritional supplement and pharmaceutical ingredients. An adequate
dietary intake of EPA and DHA has been found to help prevent inflammatory diseases, including
cardiovascular diseases, bowel diseases, cancer and arthritis (Wall et al. 2010). The nutritional
supplement DHA from Thraustochytrids (Schizochytrium sp.) has been used in foods like butter,
cheese, yoghurt and cereals (Ward et al. 2005). In addition, Thraustochytrid derived PUFAs has
found its way into aquaculture feed (Song et al. 2007a).
The marine ecosystem covers 70% of the earth surface and is the largest ecosystem in the
biosphere. It includes oceans, estuaries, mangroves, salt marsh, lagoons, coral reefs; comprising
a highly diverse ecological system. The marine environments harbour primitive microorganisms
such as cyanobacteria, to highly evolved mammals such as whales. Such a vast marine
73
biodiversity provides a major ecological niche for the isolation of different microorganisms that
produce materials of commercial interest, such as nutraceuticals (Nagano et al. 2009; Nakazawa
et al. 2012; Quilodrán et al. 2010), antibiotics (Höller et al. 2000), novel drug molecules and
inhibitors (Fenical et al. 2006), bio-surfactants (Batista et al. 2006), enzymes (Lee et al. 2006)
and oils (Li et al. 2010). Researchers are exploring marine biodiversity for novel
Thraustochytrids capable of high levels of lipid production as a feedstock for biodiesel
production (Johnson et al. 2009), high value nutraceuticals such as DHA/DPA/EPA (Gupta et al.
2012a), carotenoids (Aki et al. 2003), squalene (Li et al. 2009), novel enzymes such as lipases
(Kanchana et al. 2011) and cellulase (Nagano et al. 2011a; Taoka et al. 2009).
India and Australia both have vast marine diversity with extreme diverse flora and fauna. The
coastline comprises of headlands, promontories, rocky shores, sandy spits, barrier beaches, open
beaches, embayment, estuaries, inlets, bays, marshy land, mangroves and offshore islands (Sanil
Kumar et al. 2006). The coastlines of both countries offer exceptional marine biodiversity for the
isolation of novel Thraustochytrids suitable for various applications. Damare et al. have isolated
different strains of Thraustochytrids from Indian Ocean water columns, decaying mangrove
leaves (Dona Paula Bay, Goa) and faecal pellets of zooplankton in the Arabian Sea (Damare et
al. 2008; Kanchana et al. 2011); suggesting an abundance of Thraustochytrids along the Indian
coastline. Lee Chang and his co-workers and Gupta and his co-workers isolated several
Thraustochytrid strains from mangroves in the state of Tasmania, Queensland and Queenscliff,
Victoria, respectively (Gupta et al. 2013b; Lee Chang et al. 2012). The Indian costal water
habitats are diverse ranging from hyper saline conditions in the Gulf of Kutch to enriched
eutrophic conditions in the Sundarban delta. Whereas the marine habitats in Australia range from
the warm northern tropical waters, the subtropical central coasts, the cool temperate waters of the
74
south, and cold sub-Antarctic and Antarctic waters. Physiology of microorganisms is reported to
be influenced by the physio-chemical factors such as rainfall, salinity, dissolved oxygen levels,
temperature, pH, macronutrient levels, and carbon content (Mutanda et al. 2011; Pereira et al.
2011). These physio-chemical factors vary in India and Australia so that there is a greater chance
of isolating diverse Thraustochytrids from both the countries.
This chapter describes the isolation, identification and heterotrophic cultivation of 34
Thraustochytrids strains isolated from Indian and Australian marine waters. Indian samples were
collected from Zuari-Mandovi mangroves, Goa on the coast of Arabian Sea. Australian samples
were collected from Barwon Heads7 Victoria near the Tasman Sea. Thraustochytrid diversity
from both the Indian and Australian marine sites was compared using statistical tools including
multivariate analysis. These strains were screened for their potential use in various applications,
including (i) biodiesel, if the strains have high levels of palmitic and oleic acid, (ii) ω-3 fatty
acids, if strains have high levels of DHA/ DPA/EPA. All of the strains (34) were selected for
genetic identification and a phylogenetic tree was prepared based on 18s rRNA gene sequencing
of these strains.
7 A joint-collection trip to Barwon heads, Victoria, Australia was carried in September 2012 and help from A. Gupta, T. Thyagarajan, and A. Byreddy in collecting Australian isolates (DT1-15) is greatly acknowledged.
75
4.2. Materials and methods
4.2.1. Reagents and chemicals
All chemicals and regents used in this study were as per given in section 3.2.1. Sample
collections were carried out in sterile 50 mL falcon tubes from Tarsons (Kolkata, WB, India).
Pine pollens were harvested from local Indian trees in spring and autoclaved at 121 °C for 20
min for sterilization.
4.2.2. Site selection and sample collection
Sites which are rich in organic matter were preferred for sample collection and isolation.
Mandovi-Zuari mangrove Goa (S15°29'57.39'',E73°52'6.13'') was selected for sample collection
in India near Arabian sea (Fig 4.1a) whereas Barwon Heads, Victoria (S38°16'52.9536",
E144°29'28.197") was selected from Australia near Tasman sea (Fig 4.1b). In India, samples
were collected in April 2013 at low tide (around 3 pm) over 30-35 km stretch in mangrove areas
of Ribandar across Mandovi-Zuari mangrove complex. In Australia, samples were collected in
September 2012 after noon at low tide over a 5 km area. Soil, water, degraded leaves (around
submerged Rhizophora tress) were collected (wading with hand) at the banks of mangroves,
estuary, open sea (Arabian Sea or Tasman Sea). 25 samples from Indian and 20 samples from
Australian sites were collected in separate sterile 50 mL falcon tubes. Physical parameters such
as pH and temperature of the collection sites were recorded with pH meter and thermometer.
Antibiotic mixture (100 μL) containing PenicillinG/Streptomycin (50 mg mL-1), rifampicin (50
mg mL-1) and Nystatin (10 mg mL-1) was added into each falcon tube and samples were stored in
dry ice packs and brought to the lab for processing, along with natural sea water, within 24 h of
collection.
76
Figure 4.1. Site selection and sample collection from Mandovi-Zuari mangroves, India and
Barwon Heads, Victoria, Australia.
77
4.2.3. Isolation of Thraustochytrids from of Indian and Australian marine samples
Direct plating and baiting methods were the two protocols used for the isolation. Soil samples
were diluted 1000 times, or leaf samples were washed with sterile sea water to reduce the
chances of contamination before spreading the samples onto agar plates. Water samples were
directly spread onto agar plates. These agar plates were supplemented with an antibiotic mixture
as described above. All the samples were baited with sterile pine pollens (25-30 mg) and
incubated at 25°C for 8-10 days until colonization appeared on the periphery of pollen. After 5
days of incubation, colonization was checked daily under light microscope (Nikon Eclipse Ni U).
Once pine pollen is colonized, 100 μL of the water was taken from surface of falcon tubes and
spread carefully onto antibiotic containing agar plates. Medium for these plates were composed
of glucose (10 gL-1), yeast extract (1 gL-1), mycological peptone (1 gL-1), agar (10 gL-1), filtered
natural sea water (100 % v/v) and an antibiotic mixture. These plates were incubated at 25°C for
7-10 days. Colonies appearing on the plates were observed under microscope (40X) and their
colony morphology studied. Thraustochytrid like colonies were picked up and further streaked
onto agar plates having the above described medium composition, with 70 % natural sea water.
Thraustochytrid like strains were purified after 3-4 streaking onto agar plates containing
antibiotic mixture.
4.2.4. Strain cultivation and maintenance
Each Thraustochytrid strain was cultivated in 50 mL GPY medium containing glucose (5 gL-1),
Yeast extract (2 gL-1), mycological peptone (2 gL-1), and artificial sea water (18 gL-1 or 50 %
v/v) at pH 6.5 for 48 h. After 48 h, 5 % v/v culture was transferred in fresh medium with the
same composition and incubated at 25 °C, 150 rpm (rotary shaker) for 5 day. Strains were
78
maintained at 25 °C on GPY agar plates with the medium composition as described above in
addition to 1% agar. Plates were sub-cultured every 20 days. For long term storage, 500 μL of 48
h old culture was added into 500 μL of 80% w/v glycerol and immediately stored at -80°C.
Glycerol solution was sterilized at 121°C for 20 min. 5 day old cultures were harvested by
centrifugation at 4000 rpm for 15 min and washed twice with double distilled water to remove
traces of medium components. All centrifugation in this study were carried out at 4000 rpm for
15 min. pH of the supernatant was measured before discarding it. Pellet was freeze dried and
DCW measured gravimetrically. For freeze drying, samples were immersed in liquid nitrogen for
2-3 min and immediately set up for freeze drying for 24-48 h. Freeze dried samples were stored
at -20°C and used for screening of Thraustochytrid strains for DCW and lipid production (rich in
fatty acids for biodiesel i.e. FAB or ω-3 fatty acids).
4.2.5. Lipid extraction and FAME analysis
Fatty acids of all the Thraustochytrid strains were analysed as per protocol given in section
3.2.10. Chemotaxonomic correlation between fatty acids of all the strains was analysed (using
principal component analysis) with Multivariate software package (MVSP 3.22, Kovach
Computing Services, UK).
4.2.6. Genetic identification of Indian and Australian Thraustochytrid strains
Genomic DNA of selected Indian and Australian Thraustochytrid strains were isolated and 18S
rRNA gene was amplified and sequenced as per protocol in section 3.2.8. The resulting 18S
rRNA gene sequence was compared with known Thraustochytrids 18S rRNA gene sequences in
NCBI gene bank database using BLAST. strains sequences along with other known sequences of
Thraustochytrids were used to construct phylogenetic tree (NJ tree) using MEGA 6 software.
79
4.3. Results and discussion
4.3.1. Isolation of Thraustochytrid strains
For isolation of Thraustochytrid strains from different samples, a three tier selection procedure
was applied that included: (i) selection of a suitable cocktail of antibiotics from penicillin G,
streptomycin, rifampicin and nystatin; (ii) pine pollen baiting; and (iii) the presence of DHA in
the fatty acid profile. Identification of each strain was confirmed using 18S rDNA gene
sequencing (Fig 4.2). Penicillin G, streptomycin and rifampicin were added to reduce bacterial
contamination and nystatin to reduce fungal contamination (Wilkens et al. 2012). Samples were
dusted with sterile pine pollens to induce chemotactic movement of zoospore and subsequent
attachment of zoospores on the periphery of pine pollen. Pine pollen has been extensively used
as bait for the isolation of Thraustochytrids from marine samples (Gupta et al. 2013b; Rosa et al.
2011). Compositional analysis of pine pollen revealed significant presence of monosaccharaides
such as glucose, mannose, and xylose (4 %-10 %), polysaccharides such as starch and pectin (30
%-44 %), protein (6 %-30 %), amino acids such as glutamic acid (14 %-22 %) and flavonoids (2
%-4%) (Gupta et al. 2013b). Pectin and glutamic acid have been reported to induce a strong
chemotactic response in Thraustochytrids zoospores. During pine pollen baiting, release of these
compounds from the ventral sulcus area of pine pollen may be the reason for colonization of the
pine pollen surface by Thraustochytrids zoospores (Fan et al. 2002b). Similar explanation was
proposed by Hayakawa and co-workers for the colonization of pine pollens by zoospores of
Actinomycetes (Hayakawa et al. 1991). However in this study no contamination of
Actinomycetes was observed since we added antibiotics in the samples to avoid the growth of
other microorganisms. In most of the agar plates, no other microorganism was observed along
with Thraustochytrid colonies, indicating the robustness and accuracy of the method applied for
80
isolation of Thraustochytrids from marine samples. Based on pine pollen baiting and direct
plating methods a total of 34 Thraustochytrid strains were isolated from Indian (19 strains) and
Australian (15 strains) marine samples.
Figure 4.2. (a) Isolation method for Thraustochytrids (b) Peripheral colonization of pine pollen
by Thraustochytrids
a
b
81
4.3.2. Morphological study of Thraustochytrid strains
Most of the Thraustochytrid strains were divided in three groups based on colony colour: (i)
Orange coloured colonies; (ii) white coloured colonies; and (iii) creamy coloured colonies.
These colonies were further subdivided based on colony appearance, such as colony elevation
and colony surface. Colony elevation was flat, raised, convex, or pulvinate and surface was
either dull or glistening. Mature colonies were either spherical or irregular in shape and most of
the colonies were moderate to large in size, however some of the colonies had punctiform (Rosa
et al. 2011). During sub culturing of Thraustochytrid strains from one agar plate to another the
inoculating loop needs to be dragged from the colony to pick the colony from the agar plate.
Most of the Thraustochytrid strains (orange coloured colonies) formed small clumps in liquid
broth and if kept undisturbed for 2-5 min, cells settled down at the bottom, enabling the ready
separation of cells and broth. This attribute can be very helpful in designing energy efficient
harvesting of biomass for DHA or biodiesel production.
Thraustochytrid strains were visualized under the microscope at 40X magnification to determine
cell size, shape and reproduction method. Cell size of the strains isolated from Indian samples
ranged from 12 μm to 21 μm with average cell size of 16 μm for most of the strains. Average
cell size of Australian strains was comparatively larger than that of Indian strains, that is, 25 μm
with a range of 20 μm to 32 μm (Fig 4.3). Cell sizes of Thraustochytrium strains were larger than
Aurantiochytrium or Schizochytrium strains irrespective of location of collection of samples.
This difference in the cell size can be attributed to the existence of different reproduction method
in Thraustochytrium and Aurantiochytrium or Schizochytrium strains. Thraustochytrium are
reported to reproduce through sexual reproduction via zoospore formation (Raghukumar 2008).
In Thraustochytrium, cell size continues to increase until sporangium is fully mature to release
82
zoospores. In mature sporangium 8-10 zoospores are present. Aurantiochytrium and
Schizochytrium cells reproduce through a binary fission type of division and continue to divide at
regular intervals (Bremer 2000). This could also be the reason behind faster growth of
Aurantiochytrium or Schizochytrium strains compared with Thraustochytrium strains, which
have a lower doubling time (~5h) compared to Thraustochytrium (~10h) (Jakobsen et al. 2007;
Kimura et al. 1999).
Figure 4.3. Thraustochytrid strains from India (a-d) and Australia (e-h) marine sites (Images
were taken at 40 X magnification)
83
4.3.3. Screening of Indian and Australian Thraustochytrids for DCW, lipid, biodiesel and
ω-3 fatty acid production
After studying morphological features of the Thraustochytrid strains, all 34 strains were screened
for DCW and lipid productivity. Strains were further screened based on fatty acids composition,
as suited for biodiesel and/or ω-3 fatty acids production. The presence of DHA in the lipid is a
marker to indicate the isolated strains were Thraustochytrids. Identical cultivation parameters
were used for the comparative study of Indian and Australian Thraustochytrid so that fatty acid
profiles could be directly compared.
4.3.3.1. DCW and Lipid production
DCW production was higher in Indian strains (between 1.10 gL-1 and 3.64 gL-1) than in
Australian strains (0.67 gL-1 to 3.07 gL-1). Among Indian strains, DBTIOC-17 and DBTIOC-18
produced highest DCW, at 3.64 gL-1, followed by strain DBTIOC-1 at 3.15 gL-1 and DBTIOC-
16 at 3.04 gL-1. Lowest DCW was recorded with strain DBTIOC-20 at 1.10 gL-1. The remained
Indian strains produced DCW in range of 1.4 gL-1 to 2.6 gL-1 (Fig 4.4a). For Australian
Thraustochytrids, the maximum and minimum DCW was recorded with DT13 (3.07 gL-1) and
DT7 (0.67 gL-1), respectively. Strains DT3, DT4, DT10 and DT14 produced DCW greater than 2
gL-1. However the remaining strains produced DCW between 0.73 gL-1 and 1.8 gL-1. Higher
DCW producing strains DBTIOC-17, DBTIOC-18, DBTIOC-1 (Indian) and DT13 (Australian)
were later identified as Aurantiochytrium or Schizochytrium species. Aurantiochytrium and
Schizochytrium strains were observed to produce higher DCW as compared to Thraustochytrium
irrespective of isolation site, which is consistent with earlier reports (Burja et al. 2006; Lee
Chang et al. 2012; Yang et al. 2010).
84
In Indian Thraustochytrid strains, lipid content varied from 19 % to 50 % of DCW, with
maximum lipid content obtained from DBTIOC-1 and the minimum obtained from DBTIOC-7.
For the higher DCW producing strains, DBTIOC-4, DBTIOC-17 and DBTIOC-18, lipid content
was 31.51 %, 40.64 %, 41.97 % of DCW, respectively (Fig 4.4b). The remainder of the Indian
strains showed lipid content of around 30 % of DCW. For Australian Thraustochytrid strains, the
maximum lipid content was 51.2 % of DCW for the highest DCW producing strain DT13,
followed by 45.2 % for strain DT4. The lowest lipid content among Australian strains was 16.4
% for DT7. Lipid content was around 25% -30 % in remainder of the strains. In most of the
strains, higher DCW was an indicator of higher cellular lipid content, irrespective of isolation
sites. Therefore in most cases Thraustochytrids belonging to genus Aurantiochytrium and
Schizochytrium showed higher lipid content than Thraustochytrium.
Burja et al (2006) observed similar trend for 68 Thraustochytrid strains, isolated from Canadian
coastal sites. They reported higher DCW and lipid content with Thraustochytrium ONC-T18
(later reassign to Schizochytrium sp.) than other Thraustochytrid strains (Burja et al. 2006). Yang
and Co-workers (2010) isolated 25 Thraustochytrid strains from Taiwanese marine environment
and screened for DCW, lipid and ω-3 fatty acid production (Yang et al. 2010). They reported
higher DCW and lipid content with strain Aurantiochytrium sp. BL10 as compared to
Thraustochytrium sp. BL8. One possible reason behind higher DCW and lipid content in
Aurantiochytrium and Schizochytrium strains could be the mode of reproduction.
Thraustochytrium are stated to reproduce through zoospore formation (sexual reproduction)
whereas Schizochytrium and Aurantiochytrium reproduce through binary fission type division
(asexual reproduction) (Bongiorni et al. 2005). Growth in asexual reproduction is usually faster
than sexual reproduction. Therefore Schizochytrium and Aurantiochytrium consumes nitrogen in
85
the medium faster than Thraustochytrium, resulting in lesser time to reach nitrogen stress, which
results in faster lipid accumulation in the cell.
Figure 4.4. Comparison of (a) DCW and (b) lipid production in Indian and Australian
Thraustochytrid strains (n=2, mean±SE)
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86
4.3.3.2. Fatty acid profiling of strains for screening of FAB and ω-3 fatty acids
The presence of ω-3 fatty acids, particularly docosahexaenoic acid (DHA), in the fatty acid
profile of microorganisms is a marker of Thraustochytrids. Crythecodinium cohnii is another
microalga which synthesizes DHA. However Thraustochytrium and Crythecodinium differ in
their morphology and so can be easily differentiated under the microscope. Therefore a
microorganism isolated after applying selection pressure of antibiotic cocktail and pine pollen
baiting with DHA in their fatty acid profile is most likely to be a Thraustochytrid (Fig 4.5a). 18S
rRNA gene sequencing can be used for phylogenetic confirmation of strains after applying all
three selection pressures described earlier.
Palmitic acid (C16:0) and DHA (C22:6n3) are the major fatty acids present in the
Thraustochytrid fatty acid profile, constituting almost 70 % - 90 % of total fatty acids (TFAs),
depending on strain and culture conditions (Gupta et al. 2012a). Strains from Indian and
Australian marine biodiversity also showed a similar trend. However, in the case of Indian
Thraustochytrids, oleic acid (C18:1) was also present in significant amount (7.6 % to 30.6 % of
TFA), compared to traces amounts in the Australian Thraustochytrids (0.4 % to 4.9 % of TFA)
(Fig 4.5b). The Indian Thraustochytrids were rich in saturated fatty acids (SFAs) and
monounsaturated fatty acids (MUFAs), together constituting 51 % to 76 % of TFA. SFAs and
MUFAs are useful for biodiesel, whereas PUFA can oxidize and polymerize, causing gearing
problems. In the Indian strains, higher fatty acids for biodiesel (FAB) content was observed in
strain DBTIOC-1, DBTIOC-3, DBTIOC-17 and DBTIOC-18, whereas DHA was higher in strain
DBTIOC-4, DBTIOC-6 and DBTIOC-14. DHA content in Indian strains was modest, ranging
from 6 % to 25 % of TFA, bringing the total ω-3 fatty acid content up to 35 % of TFA. However,
in the case of Australian Thraustochytrids, the DHA content was higher, from 17 % to 31 %
87
TFA, taking total ω-3 fatty acid content up to 40 % of TFA. EPA (C20:5n3) is another important
ω-3 fatty acid with commercial applications, is present in Thraustochytrid fatty acid profile,
albeit at lower levels than DHA. EPA content is higher in Australian strains (from 3.3 % to 12.6
% of TFA) as compare to Indian strains (from 0.9 % to 3.3 % of TFA). Highest EPA content was
3.3 % (DBTIOC-21) among Indian strains and 12.6 % (DT2) among Australian strains. From the
Fig 4.5b it can be concluded that higher content of ω-3 fatty acid also translated into lower FAB
content (38 % to 63 % of TFA) in Australian strains, with the opposite for the Indian strains.
The difference in the fatty acid profile of Indian and Australian Thraustochytrids can be
attributed to the habitat and environmental conditions from where these Thraustochytrids have
been isolated. Indian Thraustochytrids were isolated from tropical wet regions of India where
summer is very hot and the average temperature can go up to 45°C, whereas Australian
Thraustochytrids were isolated from temperate regions where average temperature remains
between 15°C - 25°C. It is known that Thraustochytrids growing in colder regions (from sub-
zero to 25°C) tend to have more polyunsaturated fatty acids (PUFAs) than the Thraustochytrids
growing in tropical warm places (Lewis et al. 2001; Perveen et al. 2006). Microorganisms
growing in the these tropical areas will in turn have higher content of FAB as compared to
microorganisms isolated from colder regions (Rosa et al. 2011).
88
Figure 4.5. (a) Qualitative GC-FID of the fatty acid profile of DBTIOC-18 compared with
Schizochytrium sp. SR21, with the last two peaks showing the presence of DHA and DPA (b)
Fatty acid profiles of Indian and Australian Thraustochytrid strains showing DHA presence in all
strains
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89
4.3.3.3. Fatty acid profile as a marker for chemotaxonomic grouping of Indian and
Australian Thraustochytrid strains
Under identical culture conditions, fatty acid compositions of the strains can be used as a tool for
chemotaxonomic groupings. Fatty acids were expressed as a percentage of TFA, which was then
used as raw data for a MultiVariate Statistical Package (MVSP 3.22, Kovach Computing
Services, UK). Fatty acid profiles of the strains were clustered based on a Bray–Curtis similarity
matrix of chemotaxonomic profiles of individual strains (Fig 4.6). Farthest neighbour linkage
along with Spearman coefficient (> 0.8) was used to prepare a dendrogram representing the
similarity between strains. In the dendrogram, strains occupying closer position in a group and
having higher spearman coefficient tend be more closely related than others. The level of the
vertical lines joining two cases or clusters signifies the level of similarity between them.
Branching hierarchy and the level of similarity are the only important features of the
dendrogram. The exact order of the cases along the vertical axis is not significant. Therefore a
dendrogram can be considered as a mobile that allows the individual clusters to switch around.
Indian and Australian Thraustochytrids both occupy separate positions in the dendrogram,
reflecting the significant difference in fatty acid profiles of these two sets of Thraustochytrid
strains. Based on the Spearman coefficient being greater than 0.8, Australian Thraustochytrids
were grouped into 4 clusters (A, B, C, D), whereas Indian Thraustochytrids were grouped into 3
clusters (E, F, G) (Fig 4.6). Comparing the dendrogram (Fig 4.6) and Phylogenetic tree (Fig 4.8),
it can be concluded that both trees are closely related to each other. The fatty acid profile of a
strain is regulated by enzymes involved in their biosynthesis which in turn is regulated by genes
involved in enzyme expression and its activity. Strains possessing similar enzymatic machinery
tend to have similar fatty acid profile, thus occupying closer positions in the dendrogram. A
90
dendrogram of strains reflects the translation of phylogenetic tree for the strains. For example in
the case of Australian strains, cluster A of the dendrogram comprises of DT3, DT7, DT9 and
DT13 as similar to group 1 of the phylogenetic tree. Similarly, cluster B and group 2, cluster C,
D and group 3. This pattern is also clearly visible in the case of Indian strains. The Spearman
coefficient value of the dendrogram and boot strap value of the phylogenetic tree can be
considered as analogue in nature, determining the confidence of branching. For example, strain
DT1 and DT2 in cluster 1 or group 2 having highest Spearman coefficients (>0.9) and boot strap
values (99). A similar trend is observed for other groups and clusters. Thus the dendrogram can
be a helpful chemotaxonomic tool in the identification and grouping of new strains.
Farthest neighbour
Spearman Coefficient
DBTIOC-1DBTIOC-12/1DBTIOC-10DBTCIOC-12/2DBTIOC-20DBTIOC-19DBTIOC-8DBTIOC-13DBTIOC-17DBTIOC-18DBTIOC-21DBTIOC-3DT14DBTIOC-5DBTIOC-4DBTIOC-14DBTIOC-6DBTIOC-16DBTIOC-15DBTIOC-7DT1DT2DT6DT10DT11DT15DT4DT8DT5DT12DT3DT7DT9DT13
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Clusters
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Figure 4.6. Dendrogram of Indian and Australian Thraustochytrid strains representing similarity
and dissimilarity among strains based on fatty acid profiles
91
4.3.4. Molecular phylogeny of Indian and Australian Thraustochytrid strains
Based on growth parameters such as DCW, lipid production, ω-3 fatty acid content and fatty
acids for biodiesel, 10 Indian Thraustochytrid strains and 13 Australian strains were selected for
18S rRNA genetic identification. PCR amplification of the 18S rRNA gene gave 1.7 kb
amplicons for all the Indian and Australian strains (Fig 4.7 and Table 4.1). Gene sequencing of
these amplicons gave 800-1250 base pairs reads. The 18S rRNA sequences of these strains were
analysed with NCBI BLAST analysis, which revealed their close homology with different
Thraustochytrids species. Nucleotide sequences of all the strains were submitted to the NCBI
GenBank database (accession number KF668624-33 for Indian strains and KF682125-37 for
Australian strains). These 23 sequences together with other Thraustochytrid sequences (retrieved
from BLAST analysis) were used to construct a phylogenetic tree using MEGA 6 software. For
phylogenetic tree construction, all the sequences were aligned using CLUSTALW feature of
MEGA 6 software (Fig 4.8). The evolutionary history was inferred using the Neighbour-Joining
method (Saitou et al. 1987). The optimal tree with the sum of branch length = 3.44374300 is
shown. The percentage of replicate trees in which the associated taxa clustered together in the
bootstrap test (1000 replicates) are shown next to the branches (Felsenstein J. 1985). The tree is
drawn to scale, with branch lengths in the same units as those of the evolutionary distances used
to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-
parameter method (Kimura 1980) and are in the units of the number of base substitutions per
site. The analysis involved 42 nucleotide sequences. Codon positions included were
1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There
were a total of 625 positions in the final dataset. Evolutionary analyses were conducted in
MEGA 6 (Tamura et al. 2013).
92
Figure 4.7. PCR amplification of 18SrRNA gene (a) Indian Thraustochytrid strains (b)
Australian Thraustochytrid strains
Table 4.1. Lane number on agarose gel representing different Thraustochytrid strains
Lane No. Indian strains (a) Australian strains (b)
1 DBTIOC-1 DT-1 2 DBTIOC-4 DT-2 3 DBTIOC-5 DT-3 4 DBTIOC-6 DT-4 5 DBTIOC-13 DT-5 6 DBTIOC-14 DT-6 7 DBTIOC-16 DT-7 8 DBTIOC-17 DT-8 9 DBTIOC-18 DT-9
10 DBTIOC-21 DT-10 11 ATCC SR21 DT-11 12 ATCC 26185 DT-12 13 DT-13
a
b
93
Figure 4.8. Phylogenetic tree for Thraustochytrids isolated from Indian and Australian marine
sites (triangle-Indian Thraustochytrids, circle-Australian Thraustochytrids)9
9 Boot strap value was higher than 50 for most of the strains which reflects the confidence of the branching
94
Taxon belonging to Indian and Australian Thraustochytrids occupy different positions on the
phylogenetic tree, reflecting the wide range of diversity among Indian and Australian strains.
This diversity can be attributed to their different habitat and environmental conditions. Based on
18SrRNA gene sequencing, Australian and Indian strains were further clustered into three
subgroups in the NJ tree. Different genera of Thraustochytrids; that is, Schizochytrium and
Thraustochytrium, are represented in different subgroups. For Australian strains, Group 1
contains 5 members mainly Schizochytrium sp., whereas Group 2 comprise of 3 members mainly
Thraustochytriidae sp. Group 3 contains 5 members mainly Thraustochytrium sp. Boot strap
value of greater than 50 shows the confidence of branching on these groups. However, for Indian
Thraustochytrids, most of strains clustered in group 3 containing 8 members, which is opposite
to the trend observed for Australian Thraustochytrids. Three genera of Thraustochytrids that is,
Schizochytrium, Thraustochytrium and Aurantiochytrium are bunched in Group 3. Group 1 and 2
comprise only one member, that is, Thraustochytriidae and Thraustochytrium, respectively.
95
4.4. Conclusion
DCW, lipid production, ω-3 fatty acid content, and FAB content were the four parameters
selected to screen Indian and Australian Thraustochytrid strains for application in biodiesel
synthesis and DHA production. Based on these parameters, from 19 Indian strains, 10 strain
(DBTIOC-1, DBTIOC-4, DBTIOC-5, DBTIOC-6, DBTIOC-13 DBTIOC-14, DBTIOC-14,
DBTIOC-16, DBTIOC-17, DBTIOC-18, and DBTIOC-21) appear to have potential for either
DHA or biodiesel production. Form 15 Australian strains, 13 strains (DT1, DT2, DT3, DT4,
DT5, DT6, DT7, DT8, DT9, DT10, DT1 andDT12) have potential for DHA or biodiesel
production. Therefore, these strains were genetically identified and their phylogeny was
established comparing strains available from the NCBI database. Comparative analysis of the
fatty acid profile of these strains revealed that Indian Thraustochytrids are more suitable for
biodiesel production, since these strains had higher FAB content than the Australian
Thraustochytrids, while the Australian strains were more suitable for DHA production.
Optimization growth parameters for these strains would aid in determining which specific strains
can produce the greatest amounts of DCW and lipid, either as feedstock for biodiesel synthesis or
for ω-3 fatty acid production.
96
CHAPTER.5
5. Optimization of heterotrophic cultivation of Thraustochytrids for DCW,
lipid for biodiesel and ω-3 production using selected strains
5.1. Introduction
Thraustochytrids are well documented for their ability to produce high amount of
docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA). These omega-3 polyunsaturated
fatty acids (PUFAs) in particular docosahexaenoic acid (DHA), have gained significant attention
over recent years due to their broad range of beneficial effects on both human and animal health
(Gupta et al. 2012a). Fish oil remains primary commercial source of DHA. However faltering
fish oil supply and growing concern about oil quality and overfishing has encouraged the
industry to look for possibility of algal derived omega-3 PUFAs especially Thraustochytrids
derived DHA production (Gupta et al. 2012a). Thraustochytrid biomass is also a suitable
feedstock for biodiesel production since it contains large amount of myristic acid, palmitic acid
and some amount of oleic acid (Burja et al. 2006).
Inexpensive carbon sources such as waste glycerol from the biodiesel industry or fish industry,
sugar cane juice, molasses, beer residues, soybean cake, coconut water and sweet sorghum juice
have been investigated for lipid production with an aim of making the process commercially
viable (Gupta et al. 2012a; Pyle et al. 2008b; Quilodrán et al. 2009). The type of carbon source,
optimal quantity and input cost are crucial parameters for the design of a cost effective
fermentation protocol for the biodiesel industry. Thraustochytrids are reported to grow well in
medium with these carbon sources, without compromising yield. In fact, some reports have
shown higher DCW and lipid production than glucose using coconut water, raw glycerol or beer
residue as the carbon source (Scott et al. 2011; Unagul et al. 2007).
97
The availability and type of nitrogen source in the medium are key parameters affecting total
lipid yield and fatty acid profiles, since nitrogen levels in the medium regulates processes
involved in cell division, including protein and nucleic acid synthesis. Thraustochytrids are
reported to grow well on organic nitrogen sources such as peptone, tryptone, yeast extract, and
corn steep liquor and monosodium glutamate (Barclay 1994a, 1994b; William et al. 2005). Even
though the quantity of nitrogen needed for fermentation is less than that of carbon, the
availability of low cost nitrogen sources is still significant in lowering the costs of biofuel
production. Inorganic nitrogen sources are cheaper than their organic counterparts, but specific
nitrogen sources for cultivation remains to be determined. Nitrogen depletion in the medium
enhances lipid content at the expense of growth, so appropriate C/N ratios need to be selected in
the medium for optimal DCW and lipid production (Burja et al. 2006).
High cell density cultivation of Thraustochytrids is being applied for developing the industrial
scale processes for DHA production (Bailey et al. 2003). To achieve this, high concentration of
nutrients are added in the fermenters particularly carbon sources in batch or fed batch
cultivations. It is reported in the literature that high concentration of carbon sources may
negatively affect the cell growth and lipid accumulation (De Swaaf et al. 2003b; Ling et al. ; Wu
et al. 2005). Therefore it is recommended to have low concentration of carbon source in feeding
medium particularly initial medium (Lee Chang et al. 2013). However this may prolong
fermentation time for high DHA production Addition of high concentration of carbon sources in
the culture medium is reported to increase osmolarity of the medium apart from inducing
secretion of organic acids (e.g. malic acid, citric acid, pyruvic acid, acetic acid in the medium
(Lee Chang et al. 2013; Wu et al. 2005; Yan et al. 2013) in broth, which may not only lower the
pH of the medium but also decouples trans membrane pH gradient (Yan et al. 2013). Addition of
98
divalent cations such as calcium and magnesium can be helpful to avoid these problems.
Calcium ions can successfully form calcium salt of organic acids in the medium (Gharieb et al.
1998) thus arresting the fall in pH of the medium. These cations are reported to act as co-factors
in various enzyme reactions including protein synthesis and cell proliferation (Taha et al. 2013).
These cations also act as secondary messengers in signal transduction and play significant part in
managing stress responses (Chen et al. 2014). Chen and co-workers (2014) reported important
role of calcium ions in DCW and lipid production in Chlorella cultures under stress conditions.
Whereas Chen and co-workers (2011) also showed role of calcium ion in glycerol metabolism in
Dunaliella salina cultures under hyper and hypo osmotic stresses. Therefore it will be interesting
to study the impact of calcium and magnesium ions in DCW and lipid production in
Thraustochytrid cultivation under osmotic stress induced by the addition of higher concentration
of carbon sources.
In this chapter, ten Thraustochytrid isolates from Indian marine biodiversity were selected after
primary screening followed by further screening on different carbon and nitrogen sources with
aim of replacing glucose and yeast extract in the medium with comparatively inexpensive
nutrients. The most productive isolates were selected for further optimization. Higher
concentration of carbon and nitrogen sources was added in the medium to study the impact of
calcium and magnesium ions on glycerol uptake, DCW and lipid production in selected
Thraustochytrid strains under osmotic stress conditions.
99
5.2. Materials and method
5.2.1. Regents and chemicals
All chemicals and regents used in this study were as per details given in section 3.2.1. Calcium
carbonate and Magnesium sulphate were procured from Himedia (Mumbai, MH, India).
5.2.2. Screening of selected Indian Thraustochytrid isolates on different carbon and
nitrogen sources
One full loop (1 μL) of pure culture of the selected 10 Thraustochytrid isolates was taken from
an agar plate by semi quantitative loop and inoculated in 100 mL Erlenmeyer flasks containing
20 mL seed medium. Medium consisted of glucose 30 gL-1, yeast extract 10 gL-1, peptone 1 gL-1
and sea salt 18 gL-1 (to mimic 50 % of sea water strength). Medium used in this study was
sterilized by autoclaving at 121°C for 15 min unless otherwise mentioned. Five percent (v/v) of
48 h old inoculum was transferred into production medium (50 mL in 250 mL Erlenmeyer
flasks) having composition similar to inoculum medium except carbon or nitrogen sources.
Carbon sources (30 gL-1) such as glucose, glycerol, xylose or acetic acid and nitrogen sources
(10 gL-1) such as yeast extract (YE), corn steep liquor (CSL), ammonium sulphate (AS),
ammonium acetate (AA), ammonium chloride (AC), sodium nitrate (SN), potassium nitrate (PN)
or urea were added in production medium. Cultures were incubated at 25°C for 5 days at 150
rpm on rotary shaker for DCW production. After 5 day of growth, these cultures were harvested
by centrifugation at 4000 rpm for 15 min and washed twice with distilled water followed by
drying of the DCW at 90°C for 24 h to 48 h. DCW was measured gravimetrically. All
experiments are performed in duplicates and values are expressed as mean±SE
100
5.2.3. Effect of addition of calcium and magnesium ions on DCW and lipid production in
medium having varying concentration of glycerol and sodium nitrate in DBTIOC-1 and
DBTIOC-18
Four different media compositions were prepared to study the effect of calcium and magnesium
ions on DCW and lipid production in medium. In first medium, five different concentrations of
glycerol i.e. 30 gL-1, 60 gL-1, 90 gL-1, 120 gL-1, 150 gL-1 along with 10 gL-1 sodium nitrate were
added ( 50 ml in 250 ml Erlenmeyer flasks) to give C/N ratios of 8, 16, 24, 32, 4010. In second
medium, different concentration of both glycerol (30 gL-1, 60 gL-1, 90 gL-1, 120 gL-1, 150 gL-1)
and sodium nitrate (10 gL-1, 20 gL-1, 30 gL-1, 40 gL-1, 60 gL-1) was added to give a constant C/N
ratio of 8. Third medium was prepared by adding different concentrations of calcium carbonate11
(5 gL-1, 10 gL-1, 15 gL-1, 20 gL-1, 25 gL-1) in first medium. To study the effect of magnesium
ions on DCW and lipid production in medium, 10 gL-1 magnesium sulphate was added in third
medium. The rest of the medium composition and culture conditions were similar to those
described in section 5.2.2. Medium was sterilized by autoclaving at 106°C for 25 min. Before
harvesting culture at 5th day, 1 ml of 2N HCl was added in the cultures to solubilize residual
calcium carbonate in the medium. Cultures were centrifuged at 4000 rpm for 15 min for
harvesting and twice washed with distilled water to remove traces of residual medium and HCl.
DCW was dried at 90°C for 24 h to 48 h and DCW was measured gravimetrically. This dried
DCW was subsequently used for lipid quantification and fatty acid composition analysis.
10 C/N ratio was calculated based on formula given in section 3.2.4
11 Calcium carbonate is not soluble at this concentration in the medium however as culture grew, calcium carbonate was solubilized by forming calcium salt of organic acids. These organic acids are produced during growth.
101
5.2.4. Growth profile of DBTIOC-1 and DBTIOC-18 under optimized medium composition
For growth profile studies of both the isolates, 48 h old inoculum was transferred into production
medium having 120 gL-1 glycerol, 10 gL-1 sodium nitrate, 20 gL-1 calcium carbonate and 10 gL-1
magnesium sulphate. The rest of the medium composition and culture conditions were similar to
those described in section 5.2.2. 10 mL sample was harvested every 48 h for the analysis of
biomass production, lipid content and volumetric FAB and DHA production.
5.2.5. Glucose and glycerol estimation in medium with high performance liquid
chromatography (HPLC)
Glucose or glycerol in the medium was analysed as per protocol given in section 3.2.9.
5.2.6. Lipid extraction and FAME analysis
Fatty acids of dried biomass of Thraustochytrid strains were analysed as per protocol given in
section 3.2.10.
102
5.3. Results and discussion
5.3.1. Screening of selected Indian Thraustochytrid isolates for growth on a variety of
carbon and nitrogen sources
5.3.1.1. Effect of carbon sources on DCW and lipid production
Based on primary screening of Indian and Australian isolates, as described in Chapter 4, 10
potential Indian Thraustochytrid isolates were selected for further screening on different carbon
sources to assess their ability for DCW and lipid production. Glucose was totally consumed by
all isolates by day 5, even though there was variation in DCW and lipid production by the
isolates. Maximum DCW (8.64 gL-1) and lipid content (50.5 % of DCW) was reported with
isolate DBTIOC-18, followed by DBTIOC-1 (DCW 7.21 gL-1, lipid content 45 % of DCW) and
DBTIOC-17 (DCW 7.5 gL-1, lipid content 37 % of DCW). Other isolates including DBTIOC-4,
DBTIOC-5, DBTIOC-6 and DBTIOC-21, also produced comparable DCW (6.98 gL-1, 5.68 gL-1,
6.5 gL-1, 6.89 gL-1, respectively) and lipid content (44.5 %, 48 %, 37 %, 32 % of DCW,
respectively) (Fig 5.1). Lowest DCW (3.74 gL-1, 4.24 gL-1) was recorded with isolate DBTIOC-
13 and DBTIOC-14, respectively. Addition of glycerol as the sole carbon source in the medium
resulted in highest DCW (8.80 gL-1) and lipid content (66 % of DCW) with isolate DBTIOC-18,
followed by DBTIOC-1 (DCW 8.4 gL-1, lipid content 60.5 % of DCW) and DBTIOC-17 (DCW
8.27 gL-1, lipid content 49.5 % of DCW). Other isolates including DBTIOC-4, DBTIOC-5,
DBTIOC-6 and DBTIOC-16 also produced comparable DCW (4.77 gL-1, 6.26 gL-1, 6.59 gL-1,
7.71 gL-1, respectively) and lipid content (46 %, 65 %, 55 %, 63.5 % of DCW, respectively) (Fig
5.1). Lowest DCW (1.38 gL-1) was observed in isolate DBTIOC-21. Replacement of glucose
with glycerol resulted in higher DCW and lipid content for most of the isolates. This is in
agreement with the findings of Scott and co-workers, who reported an increase in DCW and lipid
103
content when Thraustochytrium ONC-T18 culture was fed with feeding glycerol alone, or a 50
% mix of glucose and glycerol (Scott et al. 2011). Higher DCW and lipid production with
glycerol in most of the isolates also reflects the high regulation of enzyme involved in glycerol
metabolism before entering the glycolytic pathway (Scott et al. 2011). The ability of both the
isolates to efficiently utilize glycerol provides a potential use for crude glycerol derived from
various sources (Ethier et al. 2011; Gupta et al. 2012a).
Figure 5.1. (a) DCW and (b) lipid production of Indian Thraustochytrid strains cultivated on
different carbon sources (n=2, mean±SE)
0123456789
10
DC
W (g
L-1 )
Indian Thraustochytrid strains
3 % glucose 3% xylose 3% glycerol 3% acetate
0102030405060708090
Lipi
d (%
of D
CW
)
Indian Thraustochytrid strains
3% glucose 3% xylose 3% glycerol 3% acetate
a.
b.
104
Acetate was also used as the sole carbon source in the medium for the cultivation of these
isolates. Literature suggests that acetate is converted to acetyl-CoA by acetyl-CoA synthetase
which acts as precursor for the glyoxylate and tricarboxylic acid cycles (TCA) (Perez-Garcia et
al. 2011). Use of 30 gL-1 acetate as the sole carbon source also gave comparable DCW and lipid
content. Maximum DCW (6.45 gL-1) and lipid content (54.5 % of DCW) was obtained with
isolate DBTIOC-18 followed by DBTIOC-1 (DCW 6.21 gL-1, lipid content 32 % of DCW) and
DBTIOC-4 (DCW 6.19 gL-1, lipid content 40.5 % of DCW) (fig 5.1). The lowest DCW (1.34 gL-
1) was reported with isolate DBTIOC-13. Most of the isolates in this study totally consumed the
30 gL-1 of acetate within the 5 day heterotrophic cultivation period. This contrasts with previous
studies where a low concentration of acetate (up to 10 gL-1) was recommended when acetate was
being used as sole carbon source (Perez-Garcia et al. 2011; Ratledge et al. 2001).
Addition of xylose as the sole carbon source in the medium resulted in lower DCW. Maximum
DCW was 2.57 gL-1 with DBTIOC-18, whereas another promising isolate DBTIOC-1 gave 1.16
gL-1 DCW. Lipid content also decreased in most of the isolates. These results are in agreement
with findings of Hong et al (2012) who showed little consumption of xylose with
Aurantiochytrium sp. KRS101 present in the medium containing a saccharified solution from
empty palm fruit bunches (Hong et al. 2011). Natural isolates of Thraustochytrids seems unable
to utilize xylose sugars for growth and DCW production.
105
5.3.1.2. Effect of carbon sources on FAB and DHA production
Most of the isolates produced substantial amounts of FAB and DHA on all the carbon sources
except xylose. Maximum DHA and FAB was recorded with isolate DBTIOC-18 using glucose
(3.01 gL-1 FAB, 0.75 gL-1 DHA) or glycerol (3.61 gL-1 FAB, 1.34 gL-1 DHA) as the sole carbon
source (Fig 5.2 & Table 5.1), followed by isolate DBTIOC-1, which produced 2.33 gL-1 FAB
and 0.64 gL-1 DHA with glucose or 3.98 gL-1 FAB, 1.11 gL-1 DHA with glycerol. Most of the
isolates except DBTIOC-1 and DBTIOC-21 produced higher DHA content with glycerol than
with glucose or acetate. Literature also suggests enhancement of PUFA synthesis over FAB
when glycerol is used in the medium (Chi et al. 2007b; Scott et al. 2011; Yokochi et al. 1998).
The glyoxylate pathway remains non-functional with glucose (Nagaraj et al. 2012; Palozza et al.
2009). However, glycerol is believed to activate this pathway thereby increasing malic acid level
in the cell, which enhances the supply of NADPH. Higher level of NADPH is reported to
promote PUFA synthesis, particularly DHA (Ren et al. 2009).
Figure 5.2. FAB and DHA content of Indian Thraustochytrid strains cultivated on different
carbon sources
0%10%20%30%40%50%60%70%80%90%
100%
DBTI
OC-
1 (g
luco
se)
DBTI
OC-
1 (x
ylos
e)DB
TIO
C-1
(gly
cero
l)DB
TIO
C-1
(ace
tate
)DB
TIO
C-4
(glu
cose
)DB
TIO
C-4
(xyl
ose)
DBTI
OC-
4 (g
lyce
rol)
DBTI
OC-
4 (a
ceta
te)
DBTI
OC-
5 (g
luco
se)
DBTI
OC-
5 (x
ylos
e)DB
TIO
C-5
(gly
cero
l)DB
TIO
C-5
(ace
tate
)DB
TIO
C-6
(glu
cose
)DB
TIO
C-6
(xyl
ose)
DBTI
OC-
6 (g
lyce
rol)
DBTI
OC-
6 (a
ceta
te)
DBTI
OC-
13 (g
luco
se)
DBTI
OC-
13 (x
ylos
e)DB
TIO
C-13
(gly
cero
l)DB
TIO
C-13
(ace
tate
)DB
TIO
C-14
(glu
cose
)DB
TIO
C-14
(xyl
ose)
DBTI
OC-
14 (g
lyce
rol)
DBTI
OC-
14 (a
ceta
te)
DBTI
OC-
16 (g
luco
se)
DBTI
OC-
16 (x
ylos
e)DB
TIO
C-16
(gly
cero
l)DB
TIO
C-16
(ace
tate
)DB
TIO
C-17
(glu
cose
)DB
TIO
C-17
(xyl
ose)
DBTI
OC-
17 (g
lyce
rol)
DBTI
OC-
17 (a
ceta
te)
DBTI
OC-
18 (g
luco
se)
DBTI
OC-
18 (x
ylos
e)DB
TIO
C-18
(gly
cero
l)DB
TIO
C-18
(ace
tate
)DB
TIO
C-21
(glu
cose
)DB
TIO
C-21
(xyl
ose)
DBTI
OC-
21 (g
lyce
rol)
DBTI
OC-
21 (a
ceta
te)
FAB
, DH
A c
onte
nt (%
of T
FA)
Indian Thraustochytrid strains
Others DHA FAB
106
Table 5.1. FAB and DHA content of Indian Thraustochytrid strains cultivated on different
carbon sources12
Carbon source
Fatty acids (gL-1)
DBTIOC-1
DBTIOC-4
DBTIOC-5
DBTIOC-6
DBTIOC-13
DBTIOC-14
DBTIOC-16
DBTIOC-17
DBTIOC-18
DBTIOC-21
Glucose DHA 0.64 0.52 0.51 0.35 0.14 0.35 0.13 0.35 0.75 0.32 FAB 2.33 2.07 1.72 1.65 0.82 1.26 0.94 1.99 3.01 1.54
Xylose DHA 0.08 0.03 0.01 0.01 0 0.04 0.01 0 0.01 0.12 FAB 0.23 0.43 0.44 0.66 0 0.24 0.21 0.12 0.41 0.6
Acetate DHA 0.19 0.12 0.02 0.12 0.01 0.31 0.28 0.24 0.32 0.15 FAB 2.04 2.2 1.09 0.84 0.24 0.96 2.09 1.8 2.93 0.64
Glycerol DHA 1.11 0.49 1.04 0.98 0.17 0.07 1.24 0.93 1.34 0.03 FAB 3.98 1.24 2.35 2.1 0.65 0.84 2.93 2.62 3.61 0.69
Addition of acetate to the medium appears to produce higher FAB content than DHA (Fig 5.2).
Although the use of acetate resulted in low DHA content (4.2 % to 22.4 % of TFA), DHA
production (0.12 gL-1 to 0.32 gL-1) was not low in most of the isolates, primarily because of
higher DCW and lipid production with acetate. Since acetate is a low cost carbon source it is
potentially useful for biofuel and co-product production at scale (Nie et al. 2008). Based on the
DCW, lipid, FAB and DHA production using different carbon sources, isolate DBTIOC-1 and
DBTIOC-18 were selected for subsequent trials using glycerol as the optimum carbon source.
5.3.2. Effect of nitrogen sources on DCW, lipid, FAB and DHA production in isolate
DBTIOC-1 and DBTIOC-18
The nitrogen source is reported to influence DCW and lipid productivity during heterotrophic
cultivation. Different nitrogen sources are transported and metabolized through distinct pathways
12 Values expressed in the table are the mean of duplicate samples
107
(Perez-Garcia et al. 2011; Zheng et al. 2012). Irrespective of the type of nitrogen source,
nitrogen is converted into ammonium ion before entering amino acid biosynthesis. Both isolates
showed maximum DCW (>9 gL-1 and ) and lipid production (>50 % of DCW) with yeast extract
(YE), corn steep liquor (CSL), sodium nitrate (SN), potassium nitrate (PN) (Fig 5.3).
Ammonium sulphates (AS), ammonium chloride (AC), ammonium acetate (AA) and urea, gave
lower DCW (0.41 gL-1 to 3.27 gL-1) with both isolates. However lipid content was higher in
DBTIOC-18 (39.5 % to 52 % of DCW) than in DBTIOC-1 (15.5 % to 31 % of DCW). This
reflects the contrasting behaviour of both the isolates since DBTIOC-18 belongs to
Aurantiochytrium sp. whereas DBTIOC-1 belongs to Schizochytrium sp.
Figure 5.3. (a) DCW and (b) lipid content of DBTIOC-1 and DBTIOC-18 cultivated on different
nitrogen sources, YE-yeast extract, CSL-corn steep liquor, AS-ammonium sulphate, AC-
ammonium chloride, AA-ammonium acetate, PN-potassium nitrate, SN-sodium nitrate
(mean±SE)
0
2
4
6
8
10
12
YE CSL AS AC AA PN SN Urea
DC
W (g
L-1 )
Nitrogen sources
DBTIOC-18 DBTIOC-1a.
b
010203040506070
YE CSL AS AC AA PN SN Urea
Lipi
d (%
of D
CW
)
Nitrogen source
DBTIOC-18 DBTIOC-1
108
Final pH of the supernatant varied considerably, with the lowest pH observed with ammonium
sulfate (pH 2.66) and ammonium chloride (pH 2.50) and the highest with urea (pH 8.57) with the
initial pH of the medium being 7. Literature suggests reduction in pH severely affects growth and
DCW production (Perez-Garcia et al. 2011), even though lipid content was higher than 20 % of
DCW in both the isolates (Ren et al. 2013a). Inability of the isolates to uptake nitrogen can also
induce nitrogen stress in the cell, leading to higher lipid accumulation. This could be the reason
for low DCW and high lipid content in these isolates.
Figure 5.4. FAB and DHA content of DBTIOC-1 and DBTIOC-18 cultivated on different
nitrogen sources
0102030405060708090
100
YE CSL AS AC AA PN SN Urea YE CSL AS AC AA PN SN Urea
FAB
, DH
A c
onte
nt (%
of T
FA)
Nitrogen source
Others DHA FAB
DBTIOC-18 DBTIOC-1
109
Table 5.2. FAB and DHA production of DBTIOC-1 and DBTIOC-18 cultivated on different
nitrogen sources13
Isolates Fatty acids (gL-1)
YE CSL AS AC AA PN SN Urea
DBTIOC-18
FAB 3.57 2.73 0.78 1.06 0.17 4.31 3.91 0.06
DHA 1.37 0.77 0.17 0.11 0.04 0.53 1.11 0.01
DBTIOC-1
FAB 4.18 2.65 0.21 0.17 0.31 3.62 4.27 0.16
DHA 0.82 0.94 0.04 0.02 0.08 0.49 0.84 0.05
FAB and DHA content varied depending on nitrogen source in both isolates. FAB and DHA
content were inversely related. For DBTIOC-18, YE and CSL gave higher DHA content than SN
or other nitrogen sources (Fig 5.4). Addition of YE to the medium gave the highest DHA yield
(1.37 gL-1), although SN produced a comparable amount of DHA i.e. 1.11 gL-1 (Table 5.2). For
DBTIOC-1, CSL gave the highest DHA content (20.37 % of TFA) and DHA production (0.94
gL-1) but there was no significant difference in FAB and DHA production using other nitrogen
sources, including YE or SN. Once SN was added in medium, FAB and DHA production was
4.27 gL-1 and 0.84 gL-1, respectively, and YE yielded similar results (Table 5.2). Other nitrogen
sources showed comparable DHA and FAB content, but the production of FAB and DHA was
low due to low DCW.
From the figure 5.3 and 5.4, it can be seen that YE and SN showed similar growth properties,
resulting in similar DCW, lipid content, FAB and DHA production. YE is a complex nitrogen
source and expensive relative to sodium nitrate. Therefore SN was selected as the nitrogen
source for further studies.
13 Values expressed in the table are the mean of duplicate samples
110
5.3.3. Effect of increasing carbon and nitrogen supply on DCW, lipid, FAB and DHA
production in DBTIOC-1 and DBTIOC-18
Increasing carbon supply at constant nitrogen concentration in the medium is reported to enhance
lipid accumulation in cells, resulting in higher DCW and lipid production (Ratledge 2004). In
DBTIOC-18, increasing the C/N ratio from 8 to 16 resulted in slightly increased DCW and lipid
production, from 8.97 gL-1 DCW and 64.0 % lipid to 10.18 gL-1 DCW and 64.5 % lipid,
respectively. Further increase in the C/N ratio did not translate into higher DCW or lipid content
(Table 5.3), although glycerol remained unutilized in the medium and residual glycerol in the
medium increased significantly from 28.8 gL-1 at a C/N ratio of 8, to 117.3 gL-1 with a C/N ratio
of 16. FAB and DHA production was greater than 4 gL-1 and 1 gL-1, respectively in most of the
cultures. There was no increase in FAB and DHA production with increasing carbon supply
since DCW production and lipid content remained unchanged (Table 5.3). Increasing carbon and
nitrogen supply in the medium will delay nitrogen stress, enhancing growth and later DCW
production when nitrogen is totally consumed in the medium (Ratledge 2004). However with
isolate DBTIOC-18, increasing carbon and nitrogen supply in the medium did not give any
higher DCW or lipid content. There was little increase in DCW and lipid from 8.01 gL-1 DCW
and 66.0 % lipid to 10.45 gL-1 DCW and 69 % lipid, respectively, when glycerol and sodium
nitrate supply was increased in medium from 30 gL-1 and 10 gL-1 to 60 gL-1 and 20 gL-1,
respectively. Maximum FAB and DHA production was 5.5 gL-1 and 1.22 gL-1, respectively.
Further increase in carbon and nitrogen supply did not result in higher FAB and DHA production
(Table 5.3)
Contrary to the above observation with isolate DBTIOC-18, DCW production almost doubled
from 8.43 gL-1 to 17.09 gL-1 with isolate DBTIOC-1 when the C/N ratio was raised from 8 to 16.
111
Although there was little increase in lipid content, FAB and DHA production increased
significantly from 3.76 gL-1 and 0.92 gL-1 to 7.98 gL-1 and 1.29 gL-1, respectively (Table 5.3). A
similar pattern was observed when both carbon and nitrogen supply were increased in the
medium. Although further increase in the C/N ratio or carbon and nitrogen supply together did
not translate into further increase in DCW production or lipid content, DHA production
increased from 0.92 gL-1 to 2.25 gL-1. Both isolates were unable to utilize higher concentration of
glycerol in the medium (Table 5.3).
Increase in carbon supply in the medium resulted into decrease in the medium pH to 3.7, which
could be due to the formation of organic acids such as malic acid, succinic acid and acetic acid.
Decrease in pH and formation of organic acid is reported to inhibit cell growth as it disturb trans
membrane pH gradients, internal osmotic pressure, intracellular pH, and amino acid synthesis
(Yan et al. 2013). Findings in this work are consistent with the findings of Ren and co-workers
(Ren et al. 2013a). They reported decreased DCW and lipid when the pH of the medium was
changed from 7 to 4. Literature indicates that a drop in pH has been reported to disrupt the
internal osmotic pressure, which in turn disturbs glycerol uptake by the cell since glycerol enters
the cell by diffusion due to an osmotic pressure gradient (Perez-Garcia et al. 2011). This could
explain the inability of the strains to utilize higher concentration of glycerol once the optimal pH
range is disturbed.
112
Table 5.3. DCW, lipid, FAB and DHA production in DBTIOC-1 and DBTIOC-18 cultivated on
different C/N ratios (n=2, mean±SE)
Isolates C/N ratio
Glycerol (gL-1)
Sodium nitrate (gL-1)
DCW (gL-1)
Lipid (% of DCW)
FAB (gL-1)
DHA (gL-1)
Residual glycerol (gL-1)
DBTIOC-18
8 30 10 8.97±0.01 64±1.5 4.01 0.85 0.05
16 60 10 10.18±0.36 64.5±0.5 4.44 1.19 28.9
24 90 10 10.57±0.15 61.5±1.0 4.47 1.12 57.05
32 120 10 9.87±0.39 54.5±1.0 3.64 0.94 89.55
40 150 10 9.95±0.14 65.5±2.5 4.57 1.04 117.3
8 30 10 8.01±0.49 66±5.0 5.52 1.04 0.10
8 60 20 10.45±0.57 69±1.0 5.5 1.22 27.1
8 90 30 8.21±0.93 69±4.0 4.13 0.87 58.05
8 120 40 8.84±0.26 62.5±8.5 4.5 0.84 90.12
8 150 50 9.83±1.13 47.5±4.5 3.83 1.04 119.1
DBTIOC-1
8 30 10 8.43±0.15 54.7±2.0 3.76 0.92 0.15
16 60 10 17.09±0.57 57.5±0.5 7.98 1.29 20.81
24 90 10 16.94±0.78 54.5±1.5 7.6 1.57 52.44
32 120 10 16.75±1.15 55.1±2.1 7.65 2.21 81.11
40 150 10 18.03±0.69 51±2.5 7.46 2.25 109.15
8 30 10 8.88±1.56 61.5±2.5 4.93 1.19 0.0
8 60 20 16.76±2.18 65.5±4.5 8.06 1.27 21.13
8 90 30 15.47±0.54 66±1.0 7.77 1.43 53.54
8 120 40 15.2±1.12 55±1.0 6.36 1.99 83.44
8 150 50 15.66±1.57 64.5±2.5 7.23 2.08 114.15
113
5.3.4. Effect of calcium and magnesium ions and increasing carbon supply on DCW, lipid,
FAB and DHA production in DBTIOC-1 and DBTIOC-18
Cations are reported to play an important role in numerous enzyme reactions as co-factors,
including protein synthesis, cell proliferation (Taha et al. 2013) and secondary messengers
production in signal transduction. These actions of cations not only govern cell growth but also
the lipid production in the cell (Chen et al. 2014). The cations of calcium and magnesium are
such examples. These ions play a significant role in managing stress responses and in carbon
metabolism, respectively. Therefore the effects of these ions were studied on DCW and lipid
production in DBTIOC-18 and DBTIOC-1.
5.3.4.1. Effect of calcium and magnesium ions on DCW and lipid production
Addition of increasing levels calcium carbonate and carbon supply to the medium resulted in
significant increases in DCW production in both the isolates. In isolate DBTIOC-18, DCW was
almost doubled from 8.97 gL-1 to greater than 15 gL-1 with the addition of calcium carbonate
alone or in combination of magnesium sulphate (Fig 5.5). Calcium ion is reported to increase
membrane permeability thereby enabling the faster transport of amino acids and glycerol into the
cell for growth (Kato et al. 2008). This may be the reason behind higher DCW production when
calcium salt was added in the medium. However lipid content decreased from 64 % (of DCW) to
44 % with calcium carbonate alone and to 50 % with calcium carbonate in combination of
magnesium sulphate. Increase in nonlipid DCW was observed from 36 % to 56 % (of DCW)
with calcium carbonate. With the addition of magnesium sulphate to the medium, nonlipid DCW
was decreased from 56 % to 50 % (of DCW), improving total volumetric lipid production in the
culture, indicating a direct role for magnesium ion in lipid biosynthesis, primarily in triacyl
114
glycerols (TAGs) formation. Diacylglycerol acyltransferase (DGAT1 and DGAT2) which
converts DAG into TAG in both the major pathways, Kennedy and monoacylglycerol pathways,
were reported to be significantly affected by magnesium (Taha et al. 2013).
Increasing carbon supply alone in the medium did not enhance DCW production. However,
addition of calcium carbonate only or with magnesium sulphate in the medium led to a rapid rise
in DCW production. When the C/N ratio was switched from 8 to 16 in the presence of calcium
and magnesium salts, DCW increased to 27.64 gL-1 with calcium salt alone or 29.06 gL-1 with
the mixture of calcium and magnesium salt together. Increasing carbon supply in the medium
also led to improvement in lipid content. With the addition of calcium salt in the medium, lipid
content increased to 49.5 % (of dry biomass) from 44 % with a C/N ratio 8. Lipid content further
improved (56 % of dry biomass) with addition of magnesium salt in this medium (Fig 5.5).
Maximum DCW (51.43 gL-1) and lipid content (64.5 % of dry biomass) was observed with a
C/N ratio of 32 in the presence of calcium salt, which resulted in the highest volumetric lipid
production of 33.36 gL-1. Further increase in the C/N ratio to 40 caused a fall in DCW and lipid
content. However, the addition of magnesium salt to calcium salt supplemented medium gave the
second highest DCW (46.66 gL-1) and lipid content (59 % of dry biomass) with a C/N ratio of
40. Similar trends were observed with isolate DBTIOC-1. DCW increased to 15 gL-1 from 8.43
gL-1 when calcium salt or a mixture of calcium and magnesium salts was added to the medium
having at a C/N ratio 8. However, lipid content decreased to 40 % (of dry biomass) from 54.7 %
with addition of calcium and magnesium salts. In isolate DBTIOC-1, addition of magnesium salt
to the medium, already containing calcium salt, led to increase in lipid content as compared to
addition of calcium salts alone. It is clear that addition of calcium salt to the medium promote
DCW production rich in nonlipid DCW (fig 5.5), however, addition of magnesium salt to the
115
medium will also enhance lipid content. With increasing carbon supply in medium containing
calcium salt or a mixture of calcium and magnesium salt, DCW increased sharply to 48.52 gL-1
with a C/N ratio 32, from 15 gL-1 with C/N ratio of 8. Lipid content also increased to 53 % (of
dry biomass) from 37.5 % with a C/N ratio of 8, which gave total volumetric lipid production of
25.71 gL-1, compared to 9.33 gL-1 with medium having no calcium or magnesium salts. This
clearly showed improved efficiency of cellular carbon metabolism with the influence of calcium
and magnesium salts. Further increases in the C/N ratio in medium having calcium and
magnesium salt resulted in the second highest DCW production (46.15 gL-1) and lipid content
(51.5 % of dry biomass), which gave total volumetric lipid production of 23.76 gL-1.
By increasing the carbon supply in the medium, pH of the medium decreased to 3.7, which
resulted in accumulation of residual glycerol in the medium and lower DCW production (Table
5.3). However, addition of calcium and magnesium salts in to the medium arrested the fall in pH
and so that the final pH remained around 7. Literature suggests that drop in pH is mainly due to
the excretion of organic acids in medium by the growing cells (Wu et al. 2005). These organic
acids are synthesized in excess during low aeration or under stress conditions (Yan et al. 2013).
HPLC analysis of the broth revealed the presence of organic acid such as malic acid, acetic acid,
citric acid, succinic acid in broth. Divalent cations such as calcium and magnesium can readily
form metal salts of these organic acids (Muryanto et al. 2014; Teir et al. 2007) thus arresting
drop in pH of the broth. Formation of calcium salts of organic acids also resulted into
solubilization of calcium carbonate in the broth, which is normally insoluble in broth or water.
These calcium ions can also acts as secondary messenger during stress conditions. Magnesium
ions act as cofactor for malic enzyme, which converts malic acid to pyruvate during
transdehydrogenase cycle. This cycle plays an important role during fatty acid biosynthesis by
116
supplying necessary NADPH for lipid production. Thus ensuring the optimum supply of
magnesium ions in the broth will enhance the conversion of malic acid to pyruvic acid, which
will not only help to increase cellular lipid production (a trend observed in Fig 5.5) but also
reduces secretion of malic acid into broth. An increase in concentration of calcium ions is
reported to stimulate the activity of plasma-membrane H+-ATPase activity via Ca2+/
calmodulin-dependent protein kinases under stress conditions (Shoresh et al. 2011), which will
successively promote the uptake of various nutrients such as amino acids, sugars vitamins etc
(Kato et al. 2008). Addition of calcium ions in broth also enhanced consumption of glycerol in
the medium translating in higher DCW production. Calcium ions are reported to acts as a switch
to control the intercellular glycerol level in hypertonic environments depending on the calcium
ion concentration (Chen et al. 2014). Higher concentrations of calcium ions (>10mM) enhance
intercellular glycerol by promoting the uptake of glycerol under hypertonic environments,
whereas low concentrations (<10mM) of calcium ions does the opposite (Issa 1996). Increasing
concentration of glycerol in the medium will enhance the hypertonicity of the solution thus
triggering osmotic stress. Addition of calcium ions reportedly enhanced uptake of glycerol by the
cell and subsequent utilization by the cell. Once glycerol is inside the cell, it is converted into
glyceraldehyde 3 phosphate (G3P) by G3PDH (glyceraldehyde 3 phosphate dehydrogenase)
before entering into the glycolysis pathway. Activity of G3PDH is also reported to be influenced
by intracellular calcium ions (Chen et al. 2011). Enhanced level of G3P will ensure continuous
supply of acetyl-CoA via pyruvate (O’Grady et al. 2011). Acetyl-CoA along with ample supply
of magnesium ions plays an important role in sustaining cell growth and lipid production
(Ratledge 2004).
117
Figure 5.5. Effect of calcium carbonate and magnesium sulphate on (a) DCW and (b) lipid
production in DBTIOC-1 and DBTIOC-18 culture (n=2, mean±SE)
5.3.4.2. Effect of calcium and magnesium ions on FAB and DHA production
Addition of calcium or mixture of calcium and magnesium salt in the medium resulted in
increased volumetric production of FAB and DHA. In isolate DBTIOC-18, DHA production
almost doubled to 1.66 gL-1 from 0.85 gL-1 and FAB production rose to 5.15 gL-1 from 4.01 gL-1,
when a mixture of calcium and magnesium salt was added to the medium with a C/N ratio of 8
(Table 5.4). With an increase in C/N ratio together with an increase in the concentration of
0
10
20
30
40
50
60
8 16 24 32 40 8 16 24 32 40
(DC
W (g
L-1 )
C/N ratios
Without CaCO3 With CaCO3 With CaCO3 + MgSO4
DBTIOC-18 DBTIOC-1
0102030405060708090
100
8 16 24 32 40 8 16 24 32 40
Lipi
d co
nten
t (%
of D
CW
)
C/N ratios
Without CaCO3 With CaCO3 With CaCO3 + MgSO4
DBTIOC-18 DBTIOC-1
a.
b.
118
calcium, there was a steady increase in volumetric production of both FAB and DHA. Maximum
DHA (13.31 gL-1) and FAB (16.01 gL-1) was recorded with a C/N ratio was 32. Further increase
in the C/N ratio to 40 caused a fall in FAB (14.04 gL-1) and DHA (4.74 gL-1) production.
However, addition of a mixture of calcium and magnesium salt into the medium resulted in
further increases in FAB (16.93 gL-1) and DHA (7.27 gL-1) production. Similar pattern was
observed for isolate DBTIOC-1, even though the maximum DHA (6.4 gL-1) obtained in this
isolates was less than that for DBTIOC-18 (13.31 gL-1). Volumetric DHA and FAB production
increased to 5.37 gL-1 and 16.5 gL-1, respectively, with the increased C/N ratio of 32 (Table 5.4).
Further increase in the C/N ratio led to a fall in FAB (6.59 gL-1) and DHA (2.24 gL-1)
production. However, addition of magnesium salt to the medium increased FAB (14.08 gL-1)
and DHA (6.4 gL-1) production at a C/N ratio of 40.
Table 5.4. Effect of Calcium carbonate and Magnesium sulphate on FAB and DHA production in
DBTIOC-1 and DBTIOC-1814
14 Values expressed in the table are the mean of duplicate samples
Medium
composition
Fatty acids (gL-1)
DBTIOC-18 DBTIOC-1
C/N 8
C/N 16
C/N 24
C/N 32
C/N 40
C/N 8
C/N 16
C/N 24
C/N 32
C/N 40
Without CaCO3
FAB 4.01 4.44 4.47 3.64 4.57 3.76 7.98 7.6 7.65 7.5
DHA 0.85 1.19 1.12 0.94 1.04 0.92 1.29 1.57 2.21 2.25
With
CaCO3
FAB 4.27 8.75 10.55 16.01 14.04 3.53 6.34 11.59 16.5 6.59
DHA 1.27 2.68 7.15 13.31 4.74 1.02 1.87 3.98 5.37 2.24
With CaCO3 + MgSO4
FAB 5.15 10.65 10.42 11.75 16.93 4.01 7.24 11.9 13.69 14.08
DHA 1.66 3.69 7.06 10.31 7.27 1.17 3.16 4.09 5.33 6.4
119
5.3.5. Growth profile of isolate DBTIOC-1 and DBTIOC-18 under optimized medium
composition
Growth profile of both isolates showed a short lag phase, reflecting the ability of the isolates to
quickly acclimatize in medium having significantly higher amount of glycerol. In isolate
DBTIOC-18, DCW reached 34.6 gL-1 at day 3, from 9.6 gL-1 at day 1. Glycerol concentration in
the medium decreased to 56.20 gL-1 at the end of day 3, from an initial concentration of 120 gL-1.
In this growth period, the increase in lipid content was linear and reached 35.41 % of DCW (Fig
5.6a), which translated into volumetric production of 7.45 gL-1 FAB and 3.45 gL-1 DHA. From
day 3 to 5, lipid content rose rapidly to 61.14 % of DCW, suggesting the onset of nitrogen stress
in the medium, triggering lipid biosynthesis in the cell. This is in agreement with literature,
which indicates the importance of nitrogen stress for stimulating lipid accumulation in the cell
(Ratledge 2004). DCW also increased rapidly to 51.72 gL-1, producing 16.71 gL-1 FAB and
13.41 gL-1 DHA in the culture. At the end of day 7, glycerol was almost completely consumed in
the medium, producing 59.72 gL-1 DCW and 65.38 % lipid content, which translated into
volumetric production of 21.51 gL-1 FAB and 15.15 gL-1 DHA. Isolate DBTIOC-1 also showed a
similar pattern, but DCW and volumetric DHA production was lower than for isolate DBTIOC-
18. At the end of day 3 DCW, lipid content, FAB and DHA were 24.5 gL-1, 28.13 %, 3.98 gL-1
and 1.24 gL-1, respectively (Fig 5.6). DCW almost doubled (48.14 gL-1 at 5th day) once nitrogen
was exhausted in the medium. Like isolate DBTIOC-18, nitrogen was consumed around day 3 of
cultivation for DBTIOC-1, which stimulated lipid accumulation in the cell. Lipid content rose to
52.44 % of DCW. This resulted in increased production of FAB (17.45 gL-1) and DHA (6.01 gL-
1). At the end of day 7, glycerol was almost completely consumed in the medium, producing
54.15 gL-1 DCW, 57.38 % lipid content, 20.59 gL-1 FAB and 8.18 gL-1 DHA.
120
Figure 5.6. Growth profile of isolate (a) DBTIOC-18 and (b) DBTIOC-1 at different growth
interval (n=2, mean±SE)
DCW, lipid and volumetric production of DHA, reported in this work is significantly higher than
for previous reports using shake flask modes of cultivation and to some extent comparable with
0
20
40
60
80
100
120
140
0
10
20
30
40
50
60
70
0 1 2 3 4 5 6 7
Res
idua
l gly
cero
l (gL
-1),
Lipi
d (%
of
DC
W)
DC
W, F
AB,
DH
A (g
L-1 )
Days
Biomass FABDHA Lipid contentResidual glycerol
0
20
40
60
80
100
120
140
0
10
20
30
40
50
60
70
0 1 2 3 4 5 6 7
Res
idua
l gly
cero
l (gL
-1),
Lipi
d (%
of
DC
W)
DC
W, F
AB
, DH
A (g
L-1 )
Days
Biomass FABDHA Lipid contentResidual glycerol
a.
b.
121
productivity of CSTR (continuous stirred tank reactor). Huang and co-workers (2012) used
Aurantiochytrium limacinum SR21 (Schizochytrium limacinum SR21) and reported 6.17 gL-1
DCW, 40 % lipid content and 0.25 gL-1 DHA after 7 day using 100 gL-1 glycerol. Fed batch
cultivation in CSTR significantly improved the three parameters, giving 61.76 gL-1 DCW, 65.2
% lipid content and 20.3 gL-1 DHA, after feeding 100 gL-1 glycerol, 40 gL-1 yeast extract and 40
gL-1 peptone (Huang et al. 2012). A similar study was conducted by Chi et al (2009) with
Schizochytrium limacinum SR21 in CSTR. They reported 37.9 gL-1 DCW and 6.56 gL-1 DHA
while studying the effect of shifting dissolve oxygen level (Chi et al. 2009), which is much lower
than reported in our work (DCW 59.72 gL-1, FAB 21.51 gL-1, and DHA 15.15 gL-1). Using
costly nutrients such as yeast extract and peptone in large quantity impacts the overall economics
of the process. Our work shows that high productivity can be obtained in shake flasks utilizing
low cost nutrients such as sodium nitrate and glycerol.
122
5.4 Conclusion
Indian Thraustochytrid isolate showed substantial DCW and lipid production when grown on a
range of carbon sources. Glycerol gave the higher DCW, lipid content and DHA production,
which was higher than that, obtained using glucose for most of the isolates. Isolate DBTIOC-1
and DBTIOC-18 yielded the highest DCW, FAB and DHA of the strains investigated. Screening
of growth of these two isolates on different nitrogen sources showed that sodium nitrate could
replace yeast extract. Therefore, costly medium nutrients such as glucose and yeast extract were
replaced with inexpensive nutrients such as glycerol and sodium nitrate. Addition of calcium and
magnesium salts during medium optimization enabled both of these isolates to utilize higher
concentration of glycerol for producing DCW and lipid, which translated into very high
volumetric production of FAB and DHA in shake flask cultures. The productivities achieved in
this work are higher than those reported in literature for shake flask modes of cultivation. This
partly reflects the robustness of the isolates selected, but also the potential of the process to
achieve high cell density cultivation in shake flasks or CSTR for higher volumetric production of
FAB and DHA.
123
CHAPTER.6
6. Effect of propyl gallate on the accumulation of saturated fatty acids, ω-3
fatty acids and carotenoids in Thraustochytrids
6.1. Introduction
The key regulatory steps for the fatty acid biosynthesis of oleaginous microorganisms are similar
to those of higher plants. The continuous supply of acetyl-CoA (the precursor for fatty acid
synthetase) and NADPH (which act as a reducing agent in fatty acid biosynthesis) are the two
major regulatory steps in fatty acid assimilation (Ratledge 2004). Therefore, comparative flux of
the acetyl CoA and NADPH pools between Kreb’s cycle and fatty acid synthesis regulates the
DCW and lipid accumulation in the cell, since both of these pathways share common substrate.
Shifts in this flux towards lipid biosynthesis and an enhanced supply of these two metabolites
will ensure higher lipid accumulation (Ren et al. 2009). Levels of malic acid are reported to play
an important role in NADPH supply in fatty acid biosynthesis, which in turn impacts PUFA
levels, particularly DHA production. Ren and co-workers reported significant increases in DHA
production when malic acid was added to the growth medium to increase the supply of NADPH
(Ren et al. 2009). Reports suggest that addition of reducing agents such as sodium thiosulfate or
methyl viologen can also increase NADPH pools, inactivate citrate synthase and activate the
glyoxalate shunt, which is not functional under glucose (Feng et al. 2005; Mandal et al. 2009).
All of the aforementioned changes appear to favour lipid accumulation over the Kreb’s cycle.
Sodium thiosulfate is known to exhibit oxygen scavenging activity, thus preventing lipid
degradation. When Ngangkham (2012) added sodium thiosulfate to the mixotrophic culture of
Chlorella sorokiniana, significant increases in DCW and lipid content were observed.
124
Additionally, SFA and MUFA content in the culture were substantially increased, while PUFA
levels declined (Ngangkham et al. 2012).
Propyl gallate, a well-known antioxidant used in the food industry as a preservative, is reported
to block pyruvate transport in mitochondria (Eler et al. 2009), thus leading to the accumulation
of pyruvate in the cytoplasm and enhanced acetyl-CoA and NADPH supply for lipid
biosynthesis. In addition, propyl gallate is reported to be a non-competitive inhibitor of Δ5 and
Δ6 desaturase enzymes. Inhibition of these two enzymes resulted in significant reduction in
PUFA biosynthesis in Mortierella alpine (Kawashima et al. 1996). We chose to study the effect
of propyl gallate on Thraustochytrids heterotrophic cultivation, since enhancing lipid production
and saturated fatty acid levels may help in the economic production of biodiesel in these
organisms.
In this chapter, we described results showing the potential of using Schizochytrium sp. S31 as a
candidate microbial system for biodiesel production, with concurrent production of the high
value co-products DHA and astaxanthin. We describe the impact of different growth parameters,
including the type of carbon source, the addition of propyl gallate, and the synergistic effects of
propyl gallate and increasing glycerol concentration, with the aim of maximizing production
during heterotrophic cultivation.
125
6.2. Materials and methods
6.2.1. Reagents and chemicals
All the chemicals and reagents used in this study were as per detail given in section 3.2.1. Propyl
gallate from Sigma-Aldrich (St. Louis, MO, USA) was used as a growth modulator. Methanol,
ethyl acetate, acetone (from Fischer Scientific, Waltham, MA, USA), were used for carotenoid
analysis. Carotenoid standards such as astaxanthin, zeaxanthin, canthaxanthin, res/meso-
astaxanthin, β-cryptoxanthin, echinenone were procured from CaroteNature (Ostermundigen,
Switzerland), whereas β-carotene was from Sigma-Aldrich (St. Louis, MO, USA). These
standards were used to identify and quantify the carotenoids present in Schizochytrium sp. S31.
6.2.2. Culture maintenance and DCW production
Schizochytrium sp. S31 (ATCC 20888) was procured from the American Type Culture
Collection (ATCC, USA) and maintained on GYP agar medium consisting of glucose (5 gL-1),
yeast extract (2 gL-1), peptone (2 gL-1), agar (10 gL-1) and sea salt (18 gL-1), at 25°C, and sub-
cultured every 25 days. Schizochytrium sp. S31 was inoculated in seed medium containing
glucose (30 gL-1), yeast extract (10 gL-1) and artificial seawater (18 gL-1), at 25°C for 48 h with
agitation at 150 rpm. 5 % of 48 h old culture was inoculated into production medium having
glucose or glycerol and this was cultivated at 25°C, 150 rpm on rotary shaker. After 192 h, DCW
was harvested and washed twice with water before freeze drying for 48 h. Freeze dried biomass
was stored at -20°C for subsequent work.
126
6.2.3. Effect of different concentrations of propyl gallate (PG) on growth profile, lipid
accumulation, lipid composition and carotenoid content
Selected concentrations of syringe filter sterilized propyl gallate (0.01 % w/v or 0.03 % w/v or
0.05 % w/v) were added into the production medium containing glycerol and this was cultivated
at 25°C, 150 rpm on rotary shaker. OD600nm and the DCW were measured at the interval of 24 h.
Freeze dried biomass was used for lipid and carotenoid extraction in this work.
6.2.4. Effect of different C/N ratios on DCW, lipid accumulation, lipid composition and
carotenoid content
Different C/N ratios (13, 26, 39, 52 and 65) along with 0.3 % (w/v) propyl gallate were used in
the production medium. Glycerol (3 %, 6 %, 9 %, 12 %, 15 % w/v) and yeast extract (1 % w/v)
were used as carbon and nitrogen sources, respectively. Culture conditions were similar to those
described in section 6.2.3.
6.2.5. Lipid extraction and FAME analysis
Fatty acids of all the Thraustochytrid strains were analysed as per protocol given in section
3.2.10.
6.2.6. Carotenoid extraction and quantification
Freeze dried biomass (25 mg) was suspended in 1 mL of 3M HCl and incubated at 30 °C for 40
min. Acid was removed by centrifuging at 4000 rpm for 15 min, followed by washing with
distilled water twice to removes traces of acid. Acetone (1 mL) was added into the pellet and this
was vortexed for 3 min. The orange coloured supernatant was harvested by centrifuging at 4000
127
rpm for 15 min. Extraction with acetone was repeated twice for the complete extraction of
carotenoids. The acetone extract was later analysed by RP-HPLC analysis.
6.2.7. RP-HPLC for carotenoid analysis
Carotenoids were analysed following the protocol described by (Armenta et al. 2006) with some
modification. Acetone extract (1 mL) was evaporated under a nitrogen stream, followed by the
addition of 1 mL of mobile phase A. This solution was filtered with a syringe filter (0.22 μm)
and analysed at 477 nm with RP-HPLC system (Agilent 1200 Series HPLC system with Agilent
1200 Series photodiode array detector) equipped with 5 μm Luna C18 reversed-phase column,
4.6 mm x 250 mm (Phenomenex, USA), and a Security guard column C18, 3.0 mm x 4.0 mm,
(Phenomenex, USA). The column was equilibrated with mobile phase A, consisting of methanol:
ethyl acetate: water (88:10:2, v/v/v/), for 10 min at the flow rate 0.75 mL min-1. This flow rate
was maintained for another 10 min and the mobile phase composition was changed to 2:50:48
(mobile phase B) over another 20 minutes. Flow rate was adjusted to 1.5 mL min-1 from 0.75 mL
min-1. This stage was kept for a further 10 min and then set for column equilibration with mobile
phase A for 10 min.
128
6.3. Results and discussion
6.3.1. Effect of carbon sources and propyl gallate on DCW, lipid accumulation, lipid
composition and carotenoid content of Schizochytrium sp. S31
Glucose is the most commonly used carbon source for heterotrophic cultivation of
Thraustochytrid due to its availability and preferential uptake by the cell. However, attempts
have been made to replace glucose with more economical carbon sources, including glycerol,
starch, molasses or sweet sorghum juice (Gupta et al. 2012a; Pyle et al. 2008b; Quilodrán et al.
2009). Glycerol has emerged as a potential inexpensive alternative to glucose since it is a by-
product from the biodiesel industry. Glycerol appears to be a better carbon source than glucose
for DCW production and lipid accumulation in Schizochytrium sp. S31 (Table 6.1). DCW and
Lipid content with glycerol (8.28±0.14 gL-1, 40.3±2.9 % w/w, respectively) were higher than
those produced using glucose (6.21±0.11 gL-1, 27.75±1.45 % w/w, respectively). These findings
are in agreement with the studies of Scott and co-workers, who reported an almost 5 % increase
in DCW and a 30 % increase in lipid content when glycerol was used as the primary carbon
source in Thraustochytrium ONC-T18 heterotrophic cultivation (Scott et al. 2011). Chi and co-
researchers observed a similar trend, where they reported higher DCW and lipid accumulation
with glycerol when compared to glucose (Chi et al. 2007b). However in this study an increase in
DCW (33 %) and lipid content (45 %) was greater than previous reports using glucose or
glycerol as carbon sources. Addition of propyl gallate (0.03 % w/v) into a medium containing
glycerol resulted in further improvement in both DCW production and lipid accumulation
(9.02±0.32 gL-1, 48.6±3.2 % w/w, respectively) (Table 6.1). DCW and lipid content increased by
9 % and 21 %, respectively, with the addition of propyl gallate.
129
Table 6.1. Effect of propyl gallate on DCW, lipid production and composition in Schizochytrium
sp. S31 (n=2, mean±SE)
Growth property Glucose (3%) Glycerol (3%) Glycerol (3%)
+ PG (0.03%)
DCW (gL-1) 6.21±0.11 8.28±0.14 9.02±0.32
DCW yield coefficient15 0.21 0.28 0.30
Lipid (% of DCW) 27.75 40.3±2.9 48.6±3.2
Lipid yield coefficient 0.06 0.11 0.15
TFA (% of DCW) 19.7 34.19±0.60 46.06±2.12
SFA/PUFA 0.77 0.46 1.05
Astaxanthin (μgg-1) 308.34 80.10±0.96 100.37±3.40
High DCW and lipid production with glycerol reflects a tight regulation of enzymes involved in
glycerol metabolism before entering in glycolytic pathway (Scott et al. 2011). This might be
enabling the cells to enhance the supply of acetyl-CoA or NADPH for lipid biosynthesis in
oleaginous microorganisms (Ratledge 2004). Glycerol as the carbon source appears to promote
PUFA synthesis over SFA or MUFA synthesis (Chi et al. 2007b; Scott et al. 2011; Yokochi et
al. 1998). A similar trend was observed in our work (Fig 6.1a), where PUFA content was more
than twice of SFA (68.02±1.71 and 31.10±1.71 % of TFA, respectively), giving a SFA/PUFA
ratio of 0.46. However, supplementation of glycerol medium with propyl gallate resulted in a
more than two fold increase in the SFA/PUFA ratio (from 0.46 to 1.05). As can be seen from
these results (Table 6.1) propyl gallate appears to be promoting SFA biosynthesis over PUFA.
The dark orange coloured DCW of Schizochytrium sp. S31 indicates the presence of carotenoid.
HPLC analysis of these carotenoids showed astaxanthin to be the primary carotenoid present in
15 Yield coefficient – g DCW or lipid produced by per unit gram of carbon source
130
this strain. Astaxanthin content was almost four times higher in glucose (308.34±5.44 μgg-1) than
in glycerol (80.10±0.96 μgg-1). Addition of propyl gallate in the glycerol medium resulted in
further increase in astaxanthin content (100.37±3.40 μgg-1) (Fig 6.1b). Lipid accumulation and
carotenoid biosynthesis are interrelated, since both share common acetyl-CoA precursor. Acetyl-
CoA is used in carotenoid biosynthesis via the mevalonate pathway (Somashekar et al. 2000).
Lipids are accumulated at the onset of the stationary phase; however, most of the secondary
metabolites, such as carotenoids are synthesized at the late stationary phase of the culture, where
cells are forced to utilize lipid reserves as an energy source. Somashaker and co-workers
reported high lipid accumulation with low carotenoid content, or vice versa, when they were
investigating the relationship between carotenoid and lipid accumulation in Rhodotorula gracilis
under different C/N ratio (Somashekar et al. 2000). These results are similar to our findings with
Schizochytrium S31. Use of glucose resulted in lower lipid content (27.75±1.45 % w/w) and high
astaxanthin content (308.34±5.44 μgg-1). This trend is opposite to the results with glycerol
(45.3±2.9 % lipid and 80.10±0.96 μgg-1 astaxanthin content).
Figure 6.1a. Effect of propyl gallate on fatty acid profile of Schizochytrium sp.S31 (n=2,
mean±SE)
0
10
20
30
40
50
60
Fatty
aci
ds (%
of T
FA)
Fatty acids
Glucose (3%) Glycerol (3%) Glycerol (3%) + PG (0.03%)
131
Figure 6.1b. Flasks showing the effect of propyl gallate (0.03 %) on biomass colour in
Schizochytrium sp.S31 cultures
6.3.2. Effect of concentration of propyl gallate (PG) on growth profile, lipid accumulation,
lipid composition and carotenoid content of Schizochytrium sp. S31
6.3.2.1. Effect on growth profile
Addition of different concentration of propyl gallate into the medium caused significant changes
in the normal growth profile of Schizochytrium sp. S31. The lag phase is relatively short in this
culture, indicating an ability of the strain to acclimatize in the medium quickly (Fig 6.2a & 6.2b).
The first 48 h of culture growth showed an exponential rise in OD600nm and DCW with the
maximum increase in the culture occurring with 0.01% propyl gallate in the medium. Between
48-72 h, the increase in OD600nm and DCW was minimal, showing the onset of the stationary
phase. Maximum DCW was observed with 0.03 % propyl gallate. However, further increases in
propyl gallate concentration to 0.05 % appeared inhibit the growth of the culture significantly
(2.05 gL-1 DCW at 72 h). This was also confirmed by microscopic observations (Fig 6.3). Thus,
Glycerol (3 %) Glycerol (3 %) + PG (0.03 %)
132
0.03 % appears to be the optimum concentration of propyl gallate, giving the maximum DCW
(13.20 gL-1 DCW at 72 h). Between 72-120 h, a steady drop in OD600nm and DCW was observed
in all the cultures. However after 120 h, a shift in the growth profile was observed in cultures
having 0.01 % and 0.03 % propyl gallate.
6.3.2.2 Effect on lipid accumulation and lipid composition
In the first 24 h, lipid accumulation was higher in cultures with 0.01 % propyl gallate (24.9 % of
DCW), followed by cultures having 0.03 % propyl gallate (18.1 % of DCW). Lipid accumulation
in the control culture and the culture with 0.05 % propyl gallate were similar (12.75 % and 13 %
of DCW, respectively). From 24-72 h, maximum lipid accumulation was recorded in cultures
with 0.03 % propyl gallate (57.1 % of DCW), followed by the control with 51.65 % of DCW.
Lipid content in the control culture (51.65 % of DCW) was higher than lipid content (44.23 % of
DCW) reported by Wu and co-workers in Schizochytrium sp. S31 culture (Wu et al. 2005). The
lowest lipid accumulation (26.75 % of DCW) was reported in cultures having 0.05 % propyl
gallate (Fig 6.2c). Between 72 h and 192 h, lipid content started declining, marking the onset of
the late stationary phase, followed by the death phase. Decline in the lipid reserves was faster in
cultures without propyl gallate, followed by cultures having 0.01 % propyl gallate or 0.03 %
propyl gallate. Cultures with 0.05 % propyl gallate showed a significant drop in lipid reserves
from 26.75 % at 72 h to 9.05 % of DCW at 192 h. Lipid reserves decreased from 57.1 % at 72 h
to 45.45 % of DCW at 192 h in cultures having 0.03 % propyl gallate. At the end of 192 h, lipid
content was maximum in cultures with 0.03 propyl gallate (45.45 %), followed by control (37.75
%) and 0.01 % propyl gallate (34.1 %). Nile red staining of the culture showed higher
133
fluorescence in the cultures with propyl gallate than the control as confirmed the positive effect
of propyl gallate on lipid accumulation.
Figure 6.2. Growth profile of Schizochytrium sp. S31 with glycerol under different
concentrations of propyl gallate (a) OD600nm (b) DCW (c) lipid content (d) change in SFA/PUFA
ratio (e) astaxanthin content at different time interval
134
C14:0, C15:0 and C16:0 are the major SFAs, while C22:6n3 and C22:5n6 are the major PUFAs,
present in the fatty acid profile of Schizochytrium sp. S31. SFA and PUFA biosynthesis were
inversely related in all cultures (Fig 6.2d). In the first 72 h of the cultures, SFA content increased
over time from 35-40 % at 24 h to 55-68 % of TFA at 72 h. Although SFA content increased in
all cultures up to 72 h, the increase was more prominent in cultures having 0.03 % propyl gallate
(66-68 % of TFA), followed by 0.01 % propyl gallate (62-65 % of TFA) and control (55-57 % of
TFA). SFA was consumed first followed by PUFAs under carbon starvation condition (Scott et
al. 2011). However, addition of propyl gallate in the medium reduced SFA consumption
compares to the control. An opposite trend was observed for PUFA content. Between 24 and 72
h, PUFA content dropped significantly in cultures having 0.03 % propyl gallate (from 48-50 %
to 25-27 % of TFA), followed by 0.01 % propyl gallate (46-48 % to 30-33 % of TFA), and then
control (from 55-57 % to 40-42 % of TFA).
6.3.2.3 Effect on carotenoid content
Astaxanthin is the major carotenoid present in Schizochytrium sp. S31, together with traces of
canthaxanthin, echinenone and ß-carotene. Astaxanthin production was quantified across the
growth phase to study the effect of propyl gallate on astaxanthin production (Fig 6.2e). In the
first 72 h, astaxanthin production remained low (12-14 μgg-1 at 24 h to 13-14 μgg-1 at 72 h) in all
cultures, in contrast to the observed increase in DCW and lipid content in this phase. As the
cultures moved from early stationary phase to late stationary phase, decreases in the lipid content
and DCW were observed, while the astaxanthin content started increasing from 96 h onwards.
The maximum increase in astaxanthin production was observed in cultures supplemented with
0.03% propyl gallate, with maximum production at 168 h. This trend is in agreement with the
135
finding of Carmona and co-workers (Carmona et al. 2003). They reported a sharp increase in
astaxanthin production after 96 h in Thraustochytrium CHN-1 strain. Astaxanthin production
was highest in cultures with 0.03 % propyl gallate (94.57 μgg-1), followed by 0.01 % propyl
gallate (80.52 μgg-1) and then the control (70.90 μgg-1). The inverse relation between lipid
content and astaxanthin production indicates utilization of lipid reserves for the production of
secondary metabolites (Somashekar et al. 2000) and also the conversion of ß-carotene into
astaxanthin (Carmona et al. 2003). Astaxanthin production remained low in the growth phase of
cultures having 0.05 % propyl gallate. There was no significant increase or decrease observed in
this culture (13.66 μgg-1 at 24 h to 13.73 μgg-1 at 192 h).
Figure 6.3. Microscopic images (40X) of the cultures fed with glycerol and different
concentration of propyl gallate. Figure showing the effect of gradual increase of propyl gallate
concentration on cell size and cell viability. Image of culture with concentration of 0.05 % PG
showing dead cells, reflecting the inhibitory effect of higher concentration of PG on the culture,
while rest of the images are showing healthy cells with numerous lipid bodies in the cells
6.3.2.4 Propyl gallate effect
Addition of propyl gallate to the medium resulted in a significant increase in DCW and lipid
accumulation. Continuous supply of acetyl-CoA and NADPH is necessary to achieve efficient
Without PG With PG (0.01 %) With PG (0.03 %) With PG (0.05 %)
136
lipid accumulation (Ren et al. 2009) and carotenoid production (Somashekar et al. 2000).
Acetyl-CoA is required as a precursor for fatty acid synthase and HMG-CoA synthase, whereas
NADPH acts as a reducing agent in fatty acid biosynthesis.
Figure 6.4. Mechanism of action of propyl gallate on pyruvate metabolism related to Kreb’s
cycle and transhydrogenase cycle modified from (Eler et al. 2009; Ratledge 2004). Enzymes: 1,
pyruvate decarboxylase; 2, malatedehydrogenase; 3, malic enzyme;4, pyruvate dehydrogenase;
5, citrate synthase; 6, ATP:citrate lyase; 7, citrate/malate translocase.
The carbon sources are reported to impact the acetyl-CoA supply in the cell. The acetyl–CoA
supply depends on the amount of pyruvate entering the mitochondria and converting to citrate,
which is later transported to the cytoplasm via the citrate malate pathway (Ratledge 2004). The
137
cytoplasmic level of pyruvate will also affect NADPH generation via malic acid. Propyl gallate
is reported to block the transport of pyruvate from the cytoplasm to mitochondria (Eler et al.
2009), leading to the accumulation of pyruvate in the cytoplasm (Fig 6.4). Accordingly,
enhanced pyruvate levels in the cytoplasm are metabolized by the cell to enhance the supply of
acetyl-CoA and NADPH, required for fatty acid biosynthesis. Propyl gallate is also reported to
slow the respiration in the cell (Eler et al. 2009). However, higher doses of propyl gallate (up to
2 mM) will completely block the transport of pyruvate from the cytoplasm to mitochondria,
causing cellular ATP depletion and cell death (Nakagawa et al. 1996; Nakagawa et al. 1998).
Similar effects of propyl gallate were observed in the culture of Schizochytrium sp. S31. Propyl
gallate was added to the medium at different concentrations, and these were 0.01 % w/v (0.47
mM), 0.03 % w/v (1.41 mM) and 0.05 % w/v (2.35 mM). Significant increases in DCW and lipid
accumulation were observed in cultures having propyl gallate at 0.01 % w/v (0.47 mM) and 0.03
% w/v (1.41 mM), as compared with the control. At the end of the culture, lipid content
remained high in the cells having propyl gallate in the medium. Since propyl gallate reduced the
rate of respiration, the utilization of lipid reserves in the late heterotrophic cultivation phase
remained slow in cultures supplemented with propyl gallate. Further increase in propyl gallate
concentration to 0.05 % w/v (2.35 mM) resulted in significant inhibition in DCW and lipid
accumulation (Fig 6.3). Swaaf and co-workers also observed decreased DCW and lipid
accumulation in Crypthecodinium cohnii culture when they supplemented the medium with 100
mgL-1 propyl gallate (De Swaaf et al. 2003a).
Propyl gallate is reported to be a non-competitive inhibitor of Δ5 and Δ6 desaturase enzymes.
Inhibition of these two enzymes by propyl gallate resulted in significant reduction in PUFA
biosynthesis in Mortierella alpine (Kawashima et al. 1996). Application of propyl gallate in
138
Schizochytrium sp. S31 culture resulted in decreased PUFA content and increased SFA content,
with maximum increase in cultures having 0.03 % propyl gallate, followed by 0.01 % propyl
gallate and then the control. This trend occurred across the growth phase but maximum increase
in SFA was observed at 72 h in all cultures. Reduced content of PUFA also underlines the role of
desaturases in PUFA biosynthesis in Schizochytrium sp. S31
Figure 6.5. Proposed action on propyl gallate on polyunsaturated fatty acid biosynthesis via
desaturases/elongases pathway modified from (Ratledge 2004)
139
6.3.3. Effect of different C/N ratios, at optimized propyl gallate concentration (0.03 %), on
DCW, lipid accumulation, lipid composition and carotenoid content of Schizochytrium sp.
S31
6.3.3.1 Effect of propyl gallate on DCW
Different C/N ratios were applied in the medium supplemented with an optimized amount of
propyl gallate (0.03 % w/v), to study the effect of increased supply of carbon source on DCW
and lipid accumulation in the Schizochytrium sp. S31 culture. Five C/N ratios (13, 26, 39, 52, 65)
were tested (Table 6.2). Increase in the C/N ratio resulted in a substantial increase in DCW, from
8.1 gL-1 at a C/N ratio 13, to 23.83 gL-1 at C/N ratio of 39 (Fig 6.6). However, further increase in
the ratio translated into a drop in DCW to 16.67 gL-1 at a C/N ratio 65.
Our results are in agreement with a report by Chi et. al. (2007). They reported maximum DCW
production with 8-9 % glycerol and a subsequent drop in DCW production, when glycerol
concentration was raised from 9 % to 12 % (Chi et al. 2007b; Yokochi et al. 1998). The
observed drop in DCW production may be due to excessive organic acid formation at higher
carbon levels, disturbing intracellular pH, internal osmotic pressure and amino acid synthesis.
Removal of excess acetate by converting into acetyl-CoA in Schizochytrium culture gave rise to
a significant increase in DCW (Zhao et al. 2012). Addition of 0.03 % propyl gallate at all C/N
ratios enhanced DCW production by 6-22 % (Fig 6.6). Maximum DCW (28.50 gL-1) was
produced in medium supplemented with a C/N ratio of 39 and 0.03 % propyl gallate. The DCW
yield coefficient reflects the amount of glycerol effectively converted into DCW. Increasing the
C/N ratio resulted in enhanced yield coefficient (0.305), which further increased (0.351) with
addition of 0.03% propyl gallate into the medium.
140
Figure 6.6. Effect of C/N ratios with propyl gallate (0.03 %) on DCW and lipid production in
Schizochytrium sp. S31 (n=2, mean±SE)
Table 6.2. Effect of different C/N ratios with propyl gallate (0.03 %) on DCW and lipid yields
and fatty acid ratios of SFA and PUFA
Yield coefficient (g g-1 glycerol)
C/N
ratios
Carbon source +
Propyl gallate
DCW Yield
coefficient1
Lipid yield
coefficient
SFA/PUFA
13 Glycerol (3 %) 0.27 0.10 1.11
Glycerol (3 %) + PG (0.03 %) 0.30 0.15 1.41
26 Glycerol (6 %) 0.30 0.25 1.68
Glycerol (6 %) + PG (0.03 %) 0.35 0.30 1.84
39 Glycerol (9 %) 0.26 0.21 2.18
Glycerol (9 %) + PG (0.03 %) 0.31 0.28 2.24
52 Glycerol (12 %) 0.18 0.13 3.02
Glycerol (12 %) + PG (0.03 %) 0.21 0.19 3.36
65 Glycerol (15 %) 0.11 0.08 3.75
Glycerol (15 %) + PG (0.03 %) 0.12 0.10 3.94
0 3 6 9 12 15
0
1020304050
60708090100
0
5
10
15
20
25
30
35
3 6 9 12 15
Lipi
d co
nten
t (%
of D
CW
)
DC
W, L
ipid
(gL-
1 )
C/N ratios
DCW (With PG)
DCW (Without PG)
Lipid (With PG)
Lipid (Without PG)
Lipid content (With pg)
Lipid content (Without pg)
13 26 39 52 65
141
6.3.3.2 Effect of propyl gallate on lipid accumulation and composition
When the C/N ratio was changed from 13 to 26 in control cultures, lipid yield coefficient and
lipid content reached a maximum of 0.247 and 81.05 % of DCW (Table 6.2). Increased C/N ratio
boosted the availability of carbon in the medium under nitrogen limiting condition, enabling the
cells to channel extra carbon into lipid production (Raghukumar 2008). Several reports have
shown maximum DCW and lipid production at a glycerol concentration of between 8 and 10%
(Yokochi et al. 1998). These researchers recorded the highest DCW and lipid production using 9
% glycerol for Schizochytrium SR21 culture. The highest lipid content reported in our work (79.1
% of DCW) was greater than that reported by Yokochi et al (1998) (50 % of DCW), although
culture conditions were similar. This wide gap in lipid content is probably due to different
metabolism in the respective strains used. Addition of propyl gallate in the medium resulted in
further increase in lipid production at all C/N ratios. Maximum DCW (28.5 gL-1), lipid
production (18.85 gL-1) and lipid content (87.15 % of DCW) was produced at a C/N ratio of 9 in
medium supplemented with 0.03 % propyl gallate (Fig 6.6). Production in this heterotrophic
cultivation process can be compared with the work of Kim and co-workers, who were able to
achieve 28 gL-1 DCW with 64 % lipids in the heterotrophic cultivation of Aurantiochytrium sp.
KRS101, using 8 % glucose and 1 % yeast extract (Kim et al. 2013). A subsequent increase in
the C/N ratio from 39 to 65 resulted in a reduction in lipid content, lipid production and lipid
yield coefficient. Higher lipid content in cultures grown in the presence of propyl gallate, as
compared to control, reflects an ability of propyl gallate to regulate pyruvate supply to
mitochondria in high density cultures.
Increase in the carbon supply under nitrogen stress favoured SFA biosynthesis over PUFA,
resulting in increased SFA content from 30-40 % to 70-75 % of TFA. Level of the major SFAs
142
C14:0, C15:0 and C16:0 went up while the levels of C22:5n6 and C22:6n3 decreased with
increasing carbon supply, resulting in an increase in the SFA/PUFA ratio. SFA and PUFA
production peaked at a C/N ratio of 39, with 15.46 gL-1 and 6.91 gL-1, respectively (Table 6.2).
Further increase in the carbon supply reduced SFA and PUFA production to 8.39 gL-1 and 2.47
gL-1, respectively at a C/N ratio 65, but SFA content remained high (70-75 % of TFA). The
FAMEs profile altered significantly with the addition of propyl gallate. In the medium having a
C/N ratio 13, addition of propyl gallate resulted in an about 20 % increase in SFA content. DHA
production (5.19 gL-1) in this study is comparable with that reported by Unagul et al. (2007),
who reported 6 gL-1 DHA when they fed coconut water as the carbon source for the
heterotrophic cultivation of Schizochytrium mangrovei Sk-02 (Unagul et al. 2007). DHA
production, reported by Yokochi et al. 1997 (4 gL-1), Burja et al. 2006 (4.6 gL-1), Song et al.
2007 (4.7 gL-1) (Burja et al. 2006; Song et al. 2007b; Yaguchi et al. 1997) was lower than the
production (5.19 gL-1) reported in our work.
6.3.3.3 Effect of propyl gallate on carotenoid content
Addition of propyl gallate to medium with a C/N ratio of 13 resulted in a significant increase in
astaxanthin content (100.37 μgg-1) and astaxanthin production (905.82 μgL-1) (Fig 6.7).
However, astaxanthin content dropped thereafter irrespective of C/N ratio or addition of propyl
gallate to the medium. Higher astaxanthin production (727.88 μgL-1) was recorded at a C/N ratio
of 26, while the lowest was recorded with (399.51 μgL-1) a C/N ratio of 39. Astaxanthin content
and lipid content appear to be inversely related. The lowest astaxanthin content (15.87 μgg-1)
occurred in the heterotrophic cultivation that also had the highest lipid content (87.15 % of
DCW), at a C/N ratio of 39. These findings are in agreement with the work of Somashaker and
co-workers (2000). They reported higher lipid content with low carotenoid content at different
143
C/N ratios in Rhodotorula gracilis culture (Somashekar et al. 2000). As shown in Fig 6.3e on
page no. 131, most of the astaxanthin is synthesized in the late phase of the culture, by
metabolizing lipid reserves once the carbon source is totally consumed in the medium. This is
one of the main reasons that the higher astaxanthin content occurs at a C/N ratio of 13. However,
an increase in the C/N ratio ensures the long term carbon supply in the medium, thus avoiding
starvation condition, and most of this carbon will sink into lipid biosynthesis leading to higher
accumulation of lipids under nitrogen conditions. This results in low astaxanthin biosynthesis
negatively impacting astaxanthin production in the culture.
Figure 6.7. Effect of C/N ratios with propyl gallate (0.03 %) on astaxanthin content in
Schizochytrium sp. S31 (n=2, mean±SE)
0
20
40
60
80
100
120
13 26 39 52 65
Ast
axan
thin
con
tent
(μgg
-1)
C/N ratios
With PG Without PG
144
Conclusion
In this study, we have shown that the addition of propyl gallate to medium containing glycerol
resulted in increased DCW and lipid production. Schizochytrium sp. S31 can utilize glycerol, an
inexpensive alternative carbon source to glucose, efficiently to achieve high density culture with
very high lipid content. Increased supply of the carbon source with an optimal concentration of
propyl gallate under nitrogen limiting condition resulted in significant enhancement in
production. Application of the optimized concentration of propyl gallate (0.03 % w/v) resulted in
the maximum DCW and lipid production observed in this study. Production of high value co-
products such as DHA and astaxanthin in sizable quantity may be helpful in offsetting high
biodiesel production cost from these marine microbes.
145
CHAPTER.7
7. Study of cell disruption methods and the use of response surface
methodology for the optimization of astaxanthin extraction from
Thraustochytrids
7.1. Introduction
Carotenoids such as astaxanthin and canthaxanthin are widely distributed in marine
microorganisms. They are responsible for the dark-orange pigmentation of salmon and
crustaceans (Craik et al. 1987). Astaxanthin is well known for its antioxidant properties, as it
helps to neutralize free radicals in the body generated during oxidative stress. Various reports
have shown growth inhibitory effects of astaxanthin on certain types of cancers, such as colon
and hepatic cancers (Nagaraj et al. 2012; Palozza et al. 2009). Carotenoids such as astaxanthin
help to reduce the incidence of age-related macular degeneration (Santocono et al. 2007).
Astaxanthin can also reduce UV induced cell damage by acting as a photo protectant (Guerin et
al. 2003) and is reported to be a more effective photo protectant than ß-carotene and lutein . In
addition to its human health benefits, astaxanthin is used as a natural food colorant, and feed
supplement in the fish and poultry industries.
The global carotenoid market is projected to reach $1.4 billion by 2018 (BCC 2011). While most
of the demand is expected to be met by synthetic sources, the potential for toxicity due to the
presence of synthetic byproducts has resulted in demand for naturally produced astaxanthin
(Armenta et al. 2006; Puri et al. 2012b). Recently, microorganisms such as Phaffia rhodozyma,
Haematococcus pluvialis, Chlorella zofingiensis, and Chlorella protothecoides have been
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explored as potential sources of carotenoids (Cordero et al. 2011). Some researchers have
reported the biosynthesis of astaxanthin in Thraustochytrids (Aki et al. 2003; Quilodrán et al.
2010). Improving the economic viability of complete extraction of carotenoids from
microorganisms remains an important target, one in which the cell wall is a major impediment.
Complete extraction of carotenoid requires the cell wall to be disrupted by chemical or
mechanical methods. Ultrasonication is a widely used method for cell disruption in extraction of
value added products from natural DCW. However, parameters associated with ultrasonication
need be optimized to maximise carotenoid yields. Response surface methodology is a widely
accepted statistical tool for optimizing the production of value added products such as
naringinase (Puri et al. 2010), docosahexaenoic acid (DHA) (Song et al. 2007b), biodiesel
(Huang et al. 2010), and nicotinamide (Kamble et al. 2008). Central composite design is one of
the most frequently used methods for studying the effects of variable interaction and to
determine the key factors affecting response.
In the present study, the carotenoid profile of Thraustochytrium sp. S7 is examined. The effects
of chemical and mechanical methods on cell disruption were investigated to maximizing
astaxanthin extraction yields. Response surface methodology was used to optimize
ultrasonication and maximize astaxanthin yield.
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7.2. Materials and methods
7.2.1. Reagents and Chemicals
All the chemicals and reagents used in this study were as per detail given in section 6.2.1.
Hydrochloric acid (HCl), sulphuric acid (H2SO4), citric acid, oxalic acid and acetic acid were
sourced from Sigma-Aldrich (St. Louis, MO, USA). These acids were used for cell disruption
before carotenoid extraction. Ultrasonication, bead beating, homogenization were used as
mechanical means of cell disruption
7.2.2. Isolation and molecular identification
Soil, degraded vegetation and water samples were collected in 50 ml falcon tubes from coastal
areas of New Zealand and brought back to the lab. Sterile pine pollens grains were added to the
samples and incubated for 10-15 days at 17°C. Samples were checked for colonization of the
pine pollen grains by the Thraustochytrids under the microscope. Once Thraustochytrids had
colonized the pollen, a 100 μL subsample was spread plated on an agar plate containing 13 gL-1
glucose, 0.5 gL-1 yeast extract, 0.5 gL-1 peptone, 0.5 gL-1 gelatin hydrolysate and 12.6 gL-1
artificial sea salt. Antibiotic mixture was comprised of PenicillinG/Streptomycin (50 mg mL-1),
rifampicin (50 mg mL-1) and Nystatin (10 mg mL-1), and was added to agar for the selective
growth of Thraustochytrids. These agar plates were incubated at 17°C. Once the Thraustochytrid
colonies appeared on the plate they were purified by further subsampling and streaking onto agar
plates also containing the antibiotic mixture (as described above).
For molecular identification of the isolate, 1 mL of 5 day old culture was harvested and genomic
DNA was extracted according to the guidelines described in DNeasy blood and tissue kit
(Qiagen, USA). Genomic DNA was used for PCR amplification of the 18S rRNA gene using
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primers T18S1F 5′-CAACCTGGTTGATCCTGCCAGTA-3′ and T18S5R 5′-
TCACTACGGAAACCTTGTTACGAC-3′ (Honda et al. 1999). A 25 μL PCR reaction was
setup containing 12.5 μL PCR master mix (Promega, Australia), 0.5 μL of each primer (T18S1F,
T18S5R), 1 μL genomic DNA, and 10.5 μL milliQ water. The PCR program included 3 min at
94°C for initial denaturation, 45s at 94°C for final denaturation, 30s at 64°C for annealing, 2 min
at 72°C for extension, 10 min at 72°C (final extension) and 30 cycles. The PCR product was
purified from 1 % agarose gel using QiAquick gel extraction kit (Qiagen, USA) and the mixture
containing the PCR product and primers was sent to Macrogen (South Korea) for sequencing.
The resulting 18S rRNA gene sequence was compared with known Thraustochytrid 18S rRNA
gene sequences in NCBI gene bank database using BLAST. Isolate sequences along with other
known sequences from thraustochytrids were used to construct a phylogenetic tree (NJ tree)
using MEGA 5.1 software.
7.2.3. Culture maintenance, DCW production and carotenoid profiling
The inoculum for Thraustochytrium sp. S7 was prepared in a seed medium (20 mL in 100 mL
Erlenmeyer flaks) containing 0.5 g glucose, 0.2 g yeast extract, 0.2 g peptone and 50% v/v
artificial sea water at pH 6.5 at 200C, 150 rpm. Cultures were maintained on the same medium
containing 1% agar and sub-cultured after 25 days. For DCW production, 48 h old culture was
transferred at a concentration of 5 % into the production medium (100 mL, 20oC, pH 6.5)
containing 1 g glycerol, 1 g yeast extract, 0.1 g peptone, 50% v/v artificial sea water, and
cultured for 192 h in a shake flask at 150 rpm. DCW was harvested and washed twice with
distilled water and subsequently freeze dried for 24-48 h. For carotenoid profiling,
carotenoids/pigments were directly extracted from the free dried DCW without cell disruption
149
methods. Twenty five mg of freeze dried biomass was dissolved in 1 mL acetone and vortexed
for 3 min. The coloured supernatant was separated and used in RP-HPLC analysis. All
centrifugation in this study was conducted at 4,000 rpm for 15 min at 4°C.
7.2.4. Study of growth profile and astaxanthin production
To study growth profile, 10 mL of the culture was harvested every 24 h and centrifuged followed
by freeze drying for 24-48 h. For the carotenoid study, freeze dried biomass was incubated with
3M HCl (25mg mL-1) at 30°C for 40 min. Cells were washed with water 2-3 times to remove
traces of acid, followed by the addition of acetone (25 mg mL-1) and 3 min of vortexing. This
process was repeated 2-3 times until the supernatant became colourless.
7.2.5. Effect of cell disruption methods on carotenoid yield
7.2.5.1. DMSO mediated extraction
The protocol for DMSO mediated extraction was adopted from (Sedmak et al. 1990) with some
modifications. In DMSO mediated extraction, 1 mL of DMSO (preheated to 55°C) was added to
25 mg of freeze dried biomass and kept at 55°C for 60 min undisturbed. The supernatant was
removed and stored at 15°C in the dark. This cycle was repeated 3-4 times until the DCW
became colourless. To extract carotenoids, a solvent system (2 mL) containing equal measures of
ether : water (1:1) was added to the DMSO solution at a ratio of 1:2 (DMSO : solvent system).
The extract was centrifuged at 15°C and kept at -20°C for 10 min. The lower solid DMSO layer
was discarded and the upper liquid solvent layer was transferred into a fresh centrifuge tube
followed by washing (2-3 times) to remove traces of DMSO. Carotenoid extract was dried under
nitrogen stream followed by addition of an equivalent volume of acetone and stored at -20°C.
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7.2.5.2. Acid mediated cell disruption
Freeze dried biomass samples were treated with inorganic acids such as hydrochloric acid,
sulphuric acid or organic acids such as oxalic acid, citric acid, acetic acid, or sodium bicarbonate
prior to carotenoid extraction. One mL of 3M acid was added to 25 mg of freeze dried biomass
and kept at 30°C for 40 min. However, 0.1 M sodium bicarbonate was added to 25 mg of freeze
dried biomass and kept at 40°C for 24 h. The cell suspension was washed three times with
distilled water to remove traces of acid followed by addition of 1 mL acetone.
7.2.5.3. Mechanical cell disruption
Four mechanical methods including ultrasonication for 30 min, at 20 kHz (kilohertz) for 50
cycles; homogenization at 10,500 rpm for 30 min; bead beating at 25 mg/500 μL beads (0.5 μm
size) for 30 min; or rapid freezing of biomass with liquid nitrogen followed by maceration with
mortar and pestle for 3 min, to accomplish cell disruption. Cell disruption was carried out in an
ice bath to avoid solvent evaporation or carotenoid degradation from heat generated during the
process. Coloured supernatant was harvested and stored at -20°C for RP-HPLC analysis.
7.2.5.4. RP-HPLC analysis of carotenoids
Carotenoids were extracted from freeze dried biomass and analysed with RP-HPLC with
protocol given in section 6.2.7.
7.2.6. Response surface methodology design for extraction optimization
In the ultrasonication process four variables, namely solvent/DCW ratio, power, pulse length and
time, are reported to impact carotenoid extraction from DCW (Ranjan et al. 2010). These factors
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not only govern the degree of cell lysis but also the solubility of intracellular carotenoids (Ye et
al. 2011). Therefore, the effects of these variables and their interaction on response, in this case
astaxanthin yield, were rigorously studied by central composite rotatable design. The ranges of
parameters studied are given in Table 7.1. A total of 30 extractions were carried out in this
design, each in duplicate. Out of 30 extractions, 6 extractions were conducted at the centre point,
8 at the axial point and 16 at the factorial point (α=2, k<6). The number of extractions that
needed to be carried is derived in the following equation
N = 2k + 2K + n0 ………….. (1)
N- Total number of extractions in this design, K - Number of independent variables, 2k - Number
of extractions at factorial point, 2K- Number of extractions at axial point, n0- Number of
extractions at Centre point.
The response was measured in terms of astaxanthin yield (μgg-1). Statistical analysis of
responses was performed with Design Expert 7.0.0 software (Stat-Ease Inc., USA). Analysis of
variance (ANOVA) combined with Fischer’s test were used to analyse the effect and
significance of variables on the response and their interaction on astaxanthin yield. Statistical
threshold in this experiment was having p-value equal or less than 0.05 for the interactions.
Interaction of these variables was used to construct a quadratic model for the process and to
predict the optimized conditions for maximum astaxanthin yield. Response surface curves were
constructed for the interaction between two parameters and its effect on astaxanthin yield.
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7.3. Results and discussion
7.3.1. Isolation and molecular identification of isolate
A three tier selection process was used for the isolation of thraustochytrids from marine samples.
These were the use of a specific antibiotic mixture (Wilkens et al. 2012), application of pine
pollen baiting (Gupta et al. 2013b) and confirmation of the presence of ω-3 fatty acids in the
fatty acid profile of isolates (Gupta et al. 2012a). Pine pollen is rich in carbohydrates, proteins,
amino acids and lipids and acts as a food source for thraustochytrid zoospores (Gupta et al.
2013b). Wilkens et al (2012) described a specific mixture of antibiotics, using
penicillin/streptomycin/rifampicin and the antifungal nystatin for the selective isolation of
Thraustochytrids from marine environments (Wilkens et al. 2012). During the isolation, cells
were visualized under 40X magnification using a differential interference contrast microscope to
observe their morphology. Cell size of the isolate ranged between 25 and 30 μm. FAME analysis
of the extracted lipid showed the presence of the ω-3 fatty acids DHA, DPA and EPA (30-35 %
of total fatty acid) (data not shown). For the identification of the isolate, the 18S rRNA gene was
amplified and sequenced and the resulting sequence was deposited in NCBI database (accession
number KF683340). BLAST analysis of this sequence showed its maximum similarity with
Thraustochytrium striatum. The resulting sequences after BLAST, along with the isolate
sequence, were analysed with MEGA 5.1 software. The Kimura 2-parameter model, along with
1000 bootstrap replications, was selected to construct a neighbour-joining tree (Supplementary
information S1). Boot strap values of 100 showed the confidence of the branching between
isolate and its closet relative Thraustochytrium striatum (Fig 7.1).
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Figure 7.1. Phylogenetic relationship of Thraustochytrium sp. S7 with other Thraustochytrids
7.3.2. Carotenoid profiling of Thraustochytrium sp. S7
The dark orange colour of the DCW indicated the occurrence of carotenoid biosynthesis in
Thraustochytrium sp. S7. RP-HPLC analysis of the orange coloured acetone extract revealed the
presence of different types of carotenoids including astaxanthin, canthaxanthin, echinenone and
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ß-carotene (fig 7.2). These carotenoids are eluted based on their polarity during RP-HPLC
analysis, which in turn depends on the number of oxygen atoms and keto or alcohol groups
present in the carotenoid molecule (Armenta et al. 2006). Astaxanthin, containing 4 oxygen
atoms, exhibits the highest polarity and is thus eluted first through the column, followed by
canthaxanthin, and echinenone. ß-carotene, with no oxygen atoms, is nonpolar and thus eluted
last. An unknown peak occurred between the elution of astaxanthin and canthaxanthin (7.87
min). This unknown peak could be phoenicoxanthin, given that its polarity lies between
zeaxanthin and canthaxanthin (Aki et al. 2003; Armenta et al. 2006). Relatively high quantities
of astaxanthin in the carotenoid profile indicate the efficient oxygenation of ß-carotene into
canthaxanthin via echinenone by ketolase followed by its quick hydroxylation into astaxanthin
by hydroxylase (Scaife et al. 2009).
Figure 7.2. RP-HPLC chromatogram of carotenoid profile of Thraustochytrium sp. S7
7.3.3. Study of growth profile and astaxanthin production in different growth phases of
Thraustochytrium sp. S7
Astaxanthin is the dominant constituent of the Thraustochytrium sp. S7 carotenoid profile.
Astaxanthin production and DCW were observed in the culture up to 192 h. The first 48 h of
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culture was marked by a lag phase, where cells were acclimating to the medium. There was no
increase in microbial growth/DCW or astaxanthin production observed in this phase. However,
between 48 h and 96 h, a sharp increase in OD600 nm (from 0.171±0.02 to 1.172±0.09) and DCW
(from 0.315±0.03 gL-1 to 1.774±0.02 gL-1) were visible in this growth phase (fig 7.3). The
relationship between OD600nm and DCW can be described in following equation, with R2 close to
unity.
Y = 1.3711X + 0.069 (R2 0.954)…………………………………………………………………1
Figure 7.3. Growth profile and astaxanthin production over different growth phase (n=2, mean±SE)
Maximum DCW (2.07±0.02 gL-1) was observed at 120 h while OD600nm was stationary,
indicating lipid accumulation in the cell. Astaxanthin production began to increase after 72 h
(11.73±1.31 μgg-1) and increased up to 168 h (150.85±0.26 μgg-1). Between 96 h to 120 h,
growth approached the stationary phase, and decrease in DCW was observed at 120 h,
continuing to 192 h (1.55±0.05 gL-1). This could be occurring due to the utilization of lipid
reserves by the cell in the late stationary and death phases. However, astaxanthin content
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increased sharply after 96 h. Metabolism of lipid reserves into secondary metabolites and
enhanced conversion of ß-carotene into astaxanthin are the two major driving forces behind the
increase in astaxanthin production (from 11.73±1.31 μgg-1 at 72 h to 150.85 ±0.26 μgg-1at 168 h)
during the stationary phase of the culture.
7.3.4. Effect of chemical cell disruption on astaxanthin yield
After 2-3 extractions with acetone, the supernatant became colourless, while the DCW remained
dark-orange. This contrasts with reports in the literature, which suggest that extraction should be
carried out until the entire pellet becomes colourless (Armenta et al. 2006). This demonstrates
that acetone is unable to extract all of the intracellular carotenoids without effective cell
disruption. The cell wall must be disrupted either by chemical or mechanical means to facilitate
entry of solvents into the cell to solubilize intracellular carotenoids. When the cell wall was
disrupted by ultrasonication, astaxanthin yield increased multifold over direct extraction (from
26.77±1.23 μgg-1 with direct extraction method to 156.07±4.14 μgg-1 with ultrasonication). Our
findings agree with those of Michelon and co-workers, who were able to achieve an 8-10 fold
increase in carotenoid yield via various chemical and mechanical disruption methods on freeze
dried biomass of Phaffia rhodozyma (Michelon et al. 2012). Solvents exhibiting high solvent
strength such as DMSO have been also used for the astaxanthin extraction from Phaffia
rhodozyma. The use of DMSO in this study resulted in an almost 2.5 fold increase in astaxanthin
yield (67.33±6.06 μgg-1), compared with direct extraction (26.77±1.23 μgg-1). The type of DCW
and total carotenoid content are the two crucial parameters that need to be taken into account in
extraction (Sarada et al. 2006).
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Figure 7.4. Effect of chemical cell disruption methods on astaxanthin yield
The use of acids for cell disruption is another common technique utilized prior to carotenoid
extraction with acetone. Treatment of freeze dried cells with either organic or inorganic acids
resulted in significant increases in astaxanthin yield. Yields were higher for inorganic acids than
for organic acids. Maximum yield was obtained with HCl (134.91±0.32 μgg-1), followed by
H2SO4 acid (109.36±15.16 μgg-1). The lowest yield was obtained with sodium bicarbonate
(50.49±0.16 μgg-1). Fig 7.4 shows astaxanthin yield for the various acids used. It can be
concluded that astaxanthin yield is directly proportional to acidic strength (Fig 7.4). As the acidic
strength measured by pKa value increases, cell disruption efficiency is expected to increase,
enhancing carotenoid yield (Michelon et al. 2012). Similarly to what we observed in our study,
Michelon et al. (2012) reported the highest carotenoid yield with strong acids such as HCl and
the lowest with weak acids/salt, of which sodium bicarbonate exhibited the poorest yield. HCl is
the strongest acid (pKa -7), and sodium bicarbonate the most basic (pKa 6.37). Use of other
weak acids, such as acetic acid (pKa 4.75), citric acid (pKa 5.40) and oxalic acid (pKa 4.19) also
resulted in increased astaxanthin yield compared with direct extraction (56.21±15.16 μgg-1,
0
20
40
60
80
100
120
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Directextraction
DMSO Sodiumbicarbonate
Acetic acid Citric acid Oxalic acid H2SO4 HCl
Ast
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Extraction methods
158
75.84±5.20 μgg-1, 95.24±11.46 μgg-1, respectively). These findings reiterate the importance of
cell disruption prior to solvent assisted extraction.
7.3.5. Effect of mechanical cell disruption on astaxanthin yield
While acid-mediated extraction resulted in increased astaxanthin yield, it also caused some
astaxanthin degradation, which was visible in the HPLC chromatogram (data not shown).
Different mechanical cell disruption methods such as ultrasonication, bead beating,
homogenization and maceration (rapid freezing of DCW with liquid nitrogen followed by
maceration with mortar and pestle) were used in this study. Fig 7.5 shows astaxanthin yield with
different mechanical methods of cell disruption. Application of ultrasonication in cell disruption
resulted into an almost 6-fold increase in astaxanthin yield over direct the extraction method
(from 26.77±1.23 μgg-1 with direct extraction to 156.07±4.15 μgg-1 with ultrasonication).
Astaxanthin yield with ultrasonication was highest among all the cell disruption methods tested
in this study. This demonstrates the important role of cell disruption in achieving high extraction
efficiencies. Higher degrees of cell disruption result in higher astaxanthin yields, as better cell
disruption opens up the cell wall and facilitates entry of solvents to solubilize and extract
intracellular carotenoids. Armenta et al. (2006) reported maximum carotenoid yield when using
ultrasonication for cell disruption during carotenoid extraction from freeze dried biomass of
thraustochytrids (Armenta et al. 2006). Significant increases in extraction yields of fatty acids
from freeze dried biomass of Thraustochytrids were reported using ultrasonication (Burja et al.
2007). Bead beating and ultrasonication are two major cell disruption methods being used in the
extraction of oils and high-value products (Gu et al. 2008; Lee et al. 2010; Michelon et al. 2012;
Shen et al. 2009; Valduga et al. 2009).
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Figure 7.5. Effect of mechanical cell disruption methods on astaxanthin yield (n=2, mean±SE)
Astaxanthin yield increased almost 5 fold with bead beating, rising from 26.77±1.23 μgg-1 for
direct extraction to 138.53±4.27 μgg-1 with bead beating (Fig 7.5). Bead beating to disrupt freeze
dried cells resulted in a 5 to 6 fold increase in carotenoid yield in a previous study (Sedmak,
Weerasinghe, & Jolly, 1990). While bead beating is a promising cell disruption method,
industrial scaling of this method remains a major hurdle to large scale application. Astaxanthin
yield was comparable for the remaining two methods; homogenization resulted in a yield of
66.71±2.49 μgg-1 and rapid freezing of DCW followed by maceration with mortar and pestle
resulted in 64.46±2.40 μgg-1. For both methods, the yield was greater than direct extraction by a
factor of almost 2.5.
7.3.6. Response surface methodology for extraction optimization of astaxanthin
Among the cell disruption methods employed in this study, ultrasonication achieved the
maximum carotenoid yield and caused no observable carotenoid degradation. This method was
020406080
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therefore selected for further optimization via response surface methodology. A total of 30
experiments were carried out with different combinations of variables, designed to better
understand the effect of these variables on astaxanthin yield (response). The variables selected
for this study were coded as solvent/DCW (A), power (B), pulse length (C), time (D) (Table 7.1).
Table 7.1. Range of different factors studied in response surface methodology
The combinations of variables were designed using a central composite rotatable design with
α=2 and k<6. Astaxanthin yield varied from 70.38 μgg-1 to 180.95 μgg-1. The highest astaxanthin
yield was extracted with a solvent/DCW ratio of 80 and an extraction time of 20 min, whereas
astaxanthin yield was lowest for a solvent/DCW ratio of 20 and extraction time of 15 min. These
responses underline the effect of solvent/DCW ratio and time on astaxanthin yield. The
experimental and predicted values of single extraction are given in Table 7.2.
Factor Variable Units Type Low level (-1) High level (+1)
A Solvent/DCW μL mg-1 Numeric 20 100 B Power % Numeric 20 60
C Pulse Second Numeric 10 50
D Time Minute Numeric 5 25
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Table 7.2. Astaxanthin yields from experimental and predicted methods (using model equation)
with different conditions obtained from central composite design (CCD)
Run order
Solvent /DCW
(μLmg-1)
Power (%)
Pulse (Second)
Time (Minutes)
Astaxanthin (Predicted)
(mgg-1)
Astaxanthin (Experimental)
(mgg-1) 1 60 40 30 15 164.96 152.12 2 80 50 40 10 157.68 157.80 3 40 50 20 10 113.19 108.30 4 80 30 40 10 158.18 163.14 5 60 40 30 25 161.75 165.14 6 40 30 20 10 130.61 127.48 7 80 50 20 20 162.52 158.18 8 60 40 50 15 173.41 170.15 9 80 50 20 10 148.80 151.16
10 60 40 30 15 164.96 165.41 11 60 40 10 15 159.47 160.76 12 80 30 20 20 149.38 152.12 13 80 30 20 10 143.83 143.66 14 40 30 20 20 129.81 128.64 15 40 50 40 20 120.15 119.21 16 40 30 40 20 134.87 135.59 17 60 60 30 15 133.86 131.39 18 40 30 40 10 132.33 135.63 19 60 40 30 15 164.96 166.95 20 60 40 30 15 164.96 166.14 21 100 40 30 15 138.40 136.66 22 60 40 30 5 145.49 140.12 23 60 40 30 15 164.96 169.50 24 40 50 40 10 109.44 109.79 25 40 50 20 20 120.56 118.68 26 60 20 30 15 143.61 144.10 27 20 40 30 15 70.60 70.37 28 80 30 40 20 167.07 160.91 29 60 40 30 15 164.96 169.65 30 80 50 40 20 174.74 180.95
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Multiple regression analysis was used to analyse the responses, from which a second order
polynomial equation was established (irrespective of significance of coefficients) for astaxanthin
yield, describing the interaction of variables and their effects on response.
Y1 (μgg-1) = 164.97 + 16.96 × A – 2.44 × B + 3.49 × C + 4.06 × D + 5.60 × A × B + 3.16 × A ×
C + 1.59 × A × D – 1.37 × B × C + 2.04 × B × D + 0.84 × C × D – 15.11 × A2 – 6.56 × B2 + 0.37
× C2 – 2.84 × D2 …..(2)
Y1 (μgg-1) represents astaxanthin yield and A, B, C and, D represent the coded values of
solvent/DCW, power, pulse length and time, respectively. F-test and ANOVA were used to
analyse the response surface quadratic model for astaxanthin (Table 7.3).
Table 7.3. ANOVA analysis of the quadratic model and its statistical significance
Source Sum of Squares df Mean Square F-Value p-value Model 15626.87 14 1116.205 36.27633 < 0.0001
A 6905.451 1 6905.451 224.4251 < 0.0001 B 142.3615 1 142.3615 4.626704 0.0482 C 291.5404 1 291.5404 9.474976 0.0077 D 395.4481 1 395.4481 12.85194 0.0027
AB 501.2495 1 501.2495 16.29046 0.0011 AC 159.3606 1 159.3606 5.17917 0.0380 AD 40.32187 1 40.32187 1.310449 0.2703 BC 29.88552 1 29.88552 0.97127 0.3400 BD 66.78014 1 66.78014 2.170334 0.1614 CD 11.17252 1 11.17252 0.363103 0.5558 A2 6265.745 1 6265.745 203.6348 < 0.0001 B2 1179.251 1 1179.251 38.3253 < 0.0001 C2 3.760059 1 3.760059 0.122201 0.7315 D2 220.6014 1 220.6014 7.16948 0.0172
Residual 461.5428 15 30.76952 Lack of Fit 248.1396 10 24.81396 0.581387 0.7824 Pure Error 213.4032 5 42.68063 Core Total 16088.42 29
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ANOVA analysis of the response model showed that out of 14 model terms used for establishing
the response, 9 terms, A, B, C, D, AB, AC, A2, B2, and D2, had p-value less than 0.05 and were
therefore considered significant in the process. Slight changes in these variables will cause
significant changes in response. Model terms with p-value greater than 0.1, are considered
insignificant and thus not included in the analysis. The Model F-value for astaxanthin is 36.27
(p-value <0.0001), impling that the model is significant. There is a 0.01% chance that a Model F-
value this large could occur due to noise. The lack of fit F-value of 0.58 (p-value 0.7824) implies
that the lack of fit is not significant relative to the pure error.
F-value coupled with p-value (less than 0.05) suggests that the formulated regression model is
highly significant. Statistical coefficients such as the determination coefficient (R2) and multiple
corelation coefficient (R) can be used to check the accuracy of the model. The R2 value
represents the variation in actual response by experimental variables and their interactions. A
positive outcome is for R2 to approach unity, representing better correlation between
experimental and predicted values. The R2 value for astaxanthin is 0.9713 in this model. The
adjusted R2 value (0.9445) shows that only 94.45 % of total variation could be due to
independent variables, and the remaining variations cannot be explained by the model. The
predicted R2 value of 0.8921 is in reasonable agreement with the adjusted R2 value of 0.9445.
Adeq Precision measures the signal to noise ratio. A ratio greater than 4 is desirable in the
model. The obtained ratio of 26.558 indicates an adequate signal, and this model can therefore
be used to navigate the design space.
All four variables studied in this research had p-values less than 0.05. However, only two
interactions exhibited p-values less than 0.05 below this value. This showed that not all
164
intearctions were statistically significant. 3D Response surface plots and their respective contour
plots were created to study the interaction between two variables and the effect on response.
These 3D plots were created with two variables (in their optimal range) on the X and Y- axes,
with their response on the Z-axis. Contour plot shapes can be used to predict whether the
interaction between two variables is significant or not. A circular contour plot represents
insignificant interaction between two variables, whereas an elipitcal shape indicates significant
interaction between two variables (Song et al. 2007b). Changes in the shape of the contour plot
from circular to elliptical reflects a shift in the interactions between two variables from
statistically insignificant to significant. Fig 7.6a and b (p = 0.0011 and 0.0380, respectively) are
elliptical in shape. Fig 7.6a shows the association between solvent/DCW ratio and power against
astaxanthin yield. When power was set to the minimum of 20 % (maximum power is 500 W), an
increase in solvent/DCW ratio resulted in an increase in astaxanthin yield up to a limited degree,
further increases in this ratio resulted in a consequent drop in astaxanthin yield. A similar pattern
was observed when power was set at the maximum of 60 %. Since the solvent/DCW ratio
determines the frequency of interaction between cavitation bubbles and cells (Ranjan et al.
2010), higher solvent/DCW ratios will after a certain level decrease this frequency, whereas a
low solvent/DCW ratio will increase the frequency of these interactions.. Therefore when power
was set at maximum 60 %, no significant increase in astaxanthin yield was observed. However,
when solvent/DCW ratios were set at either the minimum or maximum, increases in power up to
40 % resulted in increased astaxanthin yield. Additional increase in power did not enhance the
yield since most of the cells were already disrupted. Therefore, maximum astaxanthin is
extracted when the solvent/DCW ratio is around 60 and power around 40 %.
165
Figure 7.6. (a-b) Response surface 3D plot showing the significant interaction of solvent/DCW
ratio, with power, pulse length on response, as applied to astaxanthin yield.
Fig 7.6b demonstrates a statistically significant association between solvent/DCW and pulse
length, showing its effect on astaxanthin yield. When pulse length was set at the maximum level
of 40 or the minimum level of 10, increases in solvent/DCW resulted in increased astaxanthin
yield, after which further increases in this ratio did not enhance yield. Increases or decreases in
the pulse length did not translate into increased astaxanthin yield. The yield remains similar
when the pulse length was set at either a maximum or minimum level and so maximum pulse
length is not necessary to maximize cell disruption. Maximum response was obtained for a
solvent/DCW ratio of 60 and pulse length between 10-40 seconds. Fig 7.7 shows the correlation
between the predicted and experimental response for astaxanthin. Distribution of values around
the regression line represents the relationship between predicted and experimental responses.
Colour coding from blue to red represents the increase of response from lowest to highest. R2
approaching unity will distribute the value along the regression line, thus reducing the variation
a b
Design-Expert® Software
Astaxanthin180.949
70.378
X1 = A: Solvent/BiomassX2 = B: Power
Actual FactorsC: Pulse = 40.00D: Time = 10.00
20.00
40.00
60.00
80.00
100.00
20.00
30.00
40.00
50.00
60.00
0
42.5
85
127.5
170
Ast
axan
thin
(μg
g-1)
Solvent/CDW Pow er (%)
Design-Expert® Software
Astaxanthin180.949
70.378
X1 = A: Solvent/BiomassX2 = C: Pulse
Actual FactorsB: Power = 37.87D: Time = 10.00
20.00
40.00
60.00
80.00
100.00
10.00
20.00
30.00
40.00
50.00
60
90
120
150
180
Ast
axan
thin
(μg
g-1)
Solvent/CDW Pulse (s)
166
between predicted and actual response and increasing model accuracy. Consequently,
ultrasound-assisted treatment improved extraction efficiency of astaxanthin.
Figure 7.7. Relationship between predicted and experimental response for astaxanthin (μgg-1)
Design-Expert® SoftwareAstaxanthin
Color points by value ofAstaxanthin:
180.949
70.378
Actual
Pre
dict
ed
Predicted vs. Actual
70.00
100.00
130.00
160.00
190.00
70.38 98.02 125.66 153.31 180.95
167
7.4. Conclusion
The carotenoid profile of Thraustochytrium sp. S7 showed that astaxanthin is the major
carotenoid present in the DCW. Different methods of chemical and mechanical cell disruption
resulted in a significant inrease in astaxanthin yield. Ultrasonication was found to result in the
highest astaxanthin yield, and so response surface methodology was employed to optimize four
variables (solvent/DCW, power, pulse length and time) for maximum yield with this disruption
method. Interactions between these four variables were correlated by a polynomial equation in a
quadratic model. Optimized conditions for astaxanthin yield were found to be solvent/DCW ratio
of 71.57, power of 37.98 % (total power 500 W), pulse length of 40s and pulse time 10 min.
168
CHAPTER.8
8. Conclusions and Future Directions
8.1 Conclusions
The following conclusions are made with respect to the work carried out in this thesis:
1. Isolation and characterization of oleaginous marine yeast and its optimized
heterotrophic cultivation.
A fast growing oleaginous yeast Rhodotorula sp. strain DBTIOC-ML3 was isolated from
Pichavaram mangroves, Tamilnadu, India. This isolate showed high DCW and lipid productivity
and was found to efficiently utilize a variety of inexpensive carbon and nitrogen sources. Highest
DCW was obtained using glycerol as the carbon source, followed by glucose and xylose. In
shake flask cultures, lipid content was greater than 20 % of DCW. This isolate was found to
tolerate high concentrations of growth inhibitors, such as furfural, hydroxymethylfurfural, acetic
acid that are present in xylose rich non-detoxified liquid hydrolysate from wheat straw, without
compromising DCW and lipid production, thus eliminating the need to detoxify hydrolysates
before heterotrophic cultivation. Scale up studies in a 2L bioreactor using non-detoxified liquid
hydrolysate showed complete utilization of the carbon source within 24 h, with high DCW and
lipid productivity. Palmitic acid (C16:0) and oleic acid (C18:1) were the major fatty acids in the
lipid produced, accounting for almost 80 % of total fatty acids, which makes the extracted lipid a
suitable feedstock for biodiesel production.
169
2. Isolation and screening of Thraustochytrids from Indian and Australian biodiversity for
ω-3 fatty acid and biodiesel applications
A total of 34 Thraustochytrid strains were isolated from Indian and Australian marine samples
after applying a selection strategy. These strains were screened for ω-3 fatty acid production,
particularly DHA, and the production of fatty acids suitable for biodiesel. GC-FID analysis of the
fatty acid profile of these strains revealed a significant difference in the fatty acid composition of
Indian and Australian Thraustochytrids. FAB content (Fatty Acids for Biodiesel) were higher for
Indian Thraustochytrids than for Australian Thraustochytrids. However, ω-3 fatty acid content
was slightly higher in Australian Thraustochytrids, as compare to Indian Thraustochytrids. A
dendrogram was constructed based on fatty acid profile of these strains. These strains were
grouped in 7 clusters based on Bray–Curtis similarity matrix in the dendrogram. 10 Indian
Thraustochytrids and 13 Australian Thraustochytrids were further selected after screening and
genetically identified. These strains showed a close relationship between the dendrogram and
phylogenetic tree.
3. Optimization of heterotrophic cultivation for selected isolates of Thraustochytrid to
optimize DCW, lipid for biodiesel and ω-3 fatty acid production.
10 Indian isolates were screened on different carbon sources for DCW, lipid and DHA
production. Two isolates i.e. DBTIOC-18 and DBTIOC-1 produced the highest DCW, FAB and
DHA among 10 isolates investigated. Expensive yeast extract was replaced with low cost
nitrogen sources, particularly sodium nitrate, without compromising productivity in these two
isolates. This will in turn reduce the production cost almost 20 fold since cost of yeast extract is
around $10/kg opposite to $0.5/kg sodium nitrate. An increase in glycerol concentration alone, or
170
with added sodium nitrate, showed poor substrate utilization, resulting in lower DCW, FAB and
DHA production. However, the addition of calcium and magnesium salts to the medium
increased glycerol utilization at higher C/N ratios, resulting in substantial rises in productivity
with isolates DBTIOC-18 and DBTIOC-1. This work showed that addition of calcium and
magnesium salts in hypertonic medium can enhance DCW ad lipid production in these two
strains. Concentration of DHA reported in this work are greater than reported in literature for
shake flask cultures using related strains.
4. Effect of propyl gallate on the accumulation of saturated fatty acids, ω-3 fatty acids and
carotenoids in Thraustochytrids
Propyl gallate was shown to have contrasting effect on Schizochytrium sp. S31 for concurrent
production of FAB, DHA and astaxanthin. Different concentrations of propyl gallate were tested
in the medium to increase the productivity during heterotrophic cultivation. Medium
supplemented with 0.03 % propyl gallate showed higher DCW production, lipid accumulation
and astaxanthin production, compared with other propyl gallate concentrations. Increasing
glycerol concentration together with the addition of 0.03 % propyl gallate resulted in a multifold
rise in volumetric production of DCW, lipid, and astaxanthin production.
5. Study of cell disruption methods and the use of response surface methodology for the
optimization of astaxanthin extraction from Thraustochytrids
Astaxanthin is the predominant carotenoid, present in Thraustochytrium sp. S7. Cells disrupted
with HCl resulted in a multifold increase in astaxanthin yield. Mechanical disruption of the cells
using ultrasonication resulted in the maximum observed astaxanthin yield. Response surface
methodology was used to optimize the ultrasonication process to increase astaxanthin yields. A
171
central composite design was used to study the interaction of four variables. These were
solvent/DCW, power, pulse, time, and their effect on astaxanthin yield was examined. All of the
four variable were statistically significant (p-value <0.05), affecting astaxanthin yield. The R2
value for this quadratic model was approaching unity, indicating the accuracy and applicability
of the model. The model constructed in this study was also applicable to other strains of
Thraustochytrids, indicating the relevance of this study to efficient extraction of carotenoids
from DCW obtained from other strains of Thraustochytrids.
8.2 Future directions
Oleaginous yeast Rhodotorula sp. DBTIOC-ML3 used in this study can efficiently utilize
hemicellulosic fraction of non-detoxified liquid hydrolysate (NDLH) of acid pre-treated wheat
straw. However, different lignocellulosic DCW (LCBs) has different inhibitor profiles.
Therefore, it will be interesting to assess the DCW and lipid production on NDLH of different
LCBs. A strain of oleaginous yeast capable of growing on acidic pH medium will significantly
bring down the cost and energy associated with broth neutralization and sterilization, since at
acidic pH most microbial contaminants won’t grow and there will be no need of sterilization and
addition of calcium oxide or sodium hydroxide for neutralization. Different approaches such as
mutagenesis and gradual adaptation on acidic broth can be applied for the strain improvement.
This will help to integrate this process with ethanol production using enzymatic hydrolysate of
different LCBs, thereby producing ethanol and biodiesel using a single platform.
Thraustochytrid strains, especially Aurantiochytrium sp. DBTIOC-18 and Schizochytrium sp.
DBTIOC-1, produced higher DCW and lipid rich in FAB and DHA using glycerol as carbon
source than they produced using glucose. This indicates the potential of these two strains for
172
utilizing glycerol derived from biodiesel production or fish oil concentration for cultivation and
production of DHA. With the increasing production of biodiesel across the globe from edible or
non-edible oil or animal fat, large amount of glycerol waste is expected to be generated. To
dispose of such large amount of impure glycerol is challenging. Using this waste glycerol for the
cultivation of Thraustochytrids could be useful for the economical production of high value
DHA, with the remaining lipid being used as feedstock for biodiesel production. Therefore,
developing a large scale cultivation process with these two strains using glycerol or biodiesel
derived glycerol is an area of future investigation. The ability of these strains to utilize acetate
also provides an opportunity to explore the possibility of using acetate containing waste streams
derived from various industries, for the cultivation of Thraustochytrids.
These strains can utilize sodium nitrate in place of yeast extract without compromising
productivity and yield, which will bring down production cost. However inability of these strains
to utilize high concentration of glycerol in the medium remains a bottleneck. Therefore in
bioreactors, glycerol feeding strategy can be designed in a way to match glycerol consumption
rate with glycerol feeding rate to achieve high cell density cultivation and to avoid hypertonic
environment due to high concentration of glycerol. Supplementation of medium with calcium
and magnesium ions also helped to solve this problem. Addition of calcium and magnesium salts
to the medium led to rapid increase in DCW and lipid production under hypertonic environment.
However, the mechanism of action needs to be investigated to better understand the growth
stimulatory properties of calcium and magnesium salts in Thraustochytrid cultivation. This will
not only help to better cope with osmotic stress induced under a hypertonic environment, but also
assist in designing fed batch strategies to achieve high cell density with higher feeding rates of
the carbon source.
173
Culture conditions are reported to affect fatty acid composition in Thraustochytrids. For
example, use of glycerol instead of glucose as carbon source or incubating cultures at lower
temperature promoted biosynthesis of DHA over FAB in this work. These properties can be
exploited to design the fermentation protocol to increase DHA or FAB production depending on
our application. Downstream processing of DHA rich DCW remains another area of interest,
which needs to be explored for DHA extraction, concentration and purification from rest of the
lipid.
174
Bibliography
Ajanovic, A 2011, 'Biofuels versus food production: Does biofuels production increase food prices?', Energy, vol. 36, no. 4, pp. 2070-2076.
Aki, T, Hachida, K, Yoshinaga, M, Katai, Y, Yamasaki, T, Kawamoto, S, Kakizono, T, Maoka, T, Shigeta, S, Suzuki, O & Ono, K 2003, 'Thraustochytrid as a potential source of carotenoids', Journal of the American Oil Chemists' Society, vol. 80, no. 8, pp. 789-794.
Amin, G, Standaert, P & Verachtert, H 1984, 'Effects of metabolic inhibitors on the alcoholic fermentation by several yeasts in batch or in immobilized cell systems', Applied Microbiology and Biotechnology, vol. 19, no. 2, pp. 91-99.
Arafiles KHV, AJ, Cordero PRF, Batoon JAL, Galura FS, Leaño EM, & GR, D 2011, 'Cultural optimization of Thraustochytrids for biomass and fatty acid production', Mycosphere, vol. 2, no. 5, pp. 521-531.
Armenta, RE, Burja, A, Radianingtyas, H & Barrow, CJ 2006, 'Critical assessment of various techniques for the extraction of carotenoids and co-enzyme Q10 from the Thraustochytrid strain ONC-T18', Journal of Agricultural and Food Chemistry, vol. 54, pp. 9752-9758.
Azócar, L, Ciudad, G, Heipieper, H & Navia, R 2010, 'Biotechnological processes for biodiesel production using alternative oils', Applied Microbiology and Biotechnology, vol. 88, no. 3, pp. 621-636.
Bahnweg, G & Sparrow Jr, FK 1972, 'Aplanochytrium kerguelensis gen. nov. spec. nov., a new phycomycete from subantarctic marine waters', Archiv für Mikrobiologie, vol. 81, no. 1, pp. 45-49.
Bailey, RB, DiMasi, D, Hansen, JM, Mirrasoul, PJ, Ruecker, CM, Veeder, I, George T. , Kaneko, T & Barclay, WR 2003, 'Enhanced production of lipids containing poloyunsaturated fatty acids by very high density cultures of eukaryotic microbes in fermentors', United States Patent.
Bajpai, D & Tyagi, VK 2006, 'Biodiesel: Source, production, composition, properties and Its benefits', Journal of Oleo Science, vol. 55, no. 10, pp. 487-502.
Bajpai, PK, Bajpai, P & Ward, OP 1991, 'Optimization of production of docosahexaenoic acid (DHA) by Thraustochytrium aureum ATCC 34304', Journal of the American Oil Chemists' Society, vol. 68, no. 7, pp. 509-514.
175
Barclay, WR 1994a, 'Food product having high concentrations of omega-3 highly unsaturated fatty acids', United States Patent 5340594.
Barclay, WR 1994b, 'Process for growing Thraustochytrium and Schizochytrium using non-chloride salts to produce a micro-floral biomass having omega-3 highly unsaturated fatty acids', United States Patent 5340742.
Batista, SB, Mounteer, AH, Amorim, FR & Tótola, MR 2006, 'Isolation and characterization of biosurfactant/bioemulsifier-producing bacteria from petroleum contaminated sites', Bioresource Technology, vol. 97, no. 6, pp. 868-875.
BCC, R 2011, 'The Global Market for Carotenoids', http://www.bccresearch.com/market-research/food-and-beverage/carotenoids-global-market-fod025d.html, accessed on 20th june 2014).
Bhosale, P 2004, 'Environmental and cultural stimulants in the production of carotenoids from microorganisms', Applied Microbiology and Biotechnology, vol. 63, no. 4, pp. 351-361.
Bollmeier, WS & Sprague, S 1989, 'Aquatic Species Program, Annual Report '.
Bongiorni, L, Jain, R, Raghukumar, S & Aggarwal, RK 2005, 'Thraustochytrium gaertnerium sp. nov.: A new Thraustochytrid stramenopilan protist from mangroves of Goa, India', Protist, vol. 156, no. 3, pp. 303-315.
Bowles, RD, Hunt, AE, Bremer, GB, Duchars, MG & Eaton, RA 1999, 'Long-chain n-3 polyunsaturated fatty acid production by members of the marine protistan group the Thraustochytrids: Screening of isolates and optimisation of docosahexaenoic acid production', Journal of Biotechnology, vol. 70, no. 1-3, pp. 193-202.
Brahamsha, B 1996, 'A genetic manipulation system for oceanic cyanobacteria of the genus Synechococcus', Applied and Environmental Microbiology, vol. 62, no. 5, pp. 1747-1751.
Braunwald, T, Schwemmlein, L, Graeff-Hönninger, S, French, W, Hernandez, R, Holmes, W & Claupein, W 2013, 'Effect of different C/N ratios on carotenoid and lipid production by Rhodotorula glutinis', Applied Microbiology and Biotechnology, vol. 97, no. 14, pp. 6581-6588.
Bremer, G 2000, 'Isolation and culture of Thraustochytrids. Marine mycology — a practical approach Hong Kong', Fungal Diversity, vol. 4, pp. 49-61.
176
Brennan, L & Owende, P 2010, 'Biofuels from microalgae-A review of technologies for production, processing, and extractions of biofuels and co-products', Renewable and Sustainable Energy Reviews, vol. 14, no. 2, pp. 557-577.
Burja, AM, Armenta, RE, Radianingtyas, H & Barrow, CJ 2007, 'Evaluation of fatty acid extraction methods for Thraustochytrium sp. ONC-T18', Journal of Agricultural and Food Chemistry, vol. 55, no. 12, pp. 4795-4801.
Burja, AM, Radianingtyas, H, Windust, A & Barrow, CJ 2006, 'Isolation and characterization of polyunsaturated fatty acid producing Thraustochytrium species: Screening of strains and optimization of omega-3 production', Applied Microbiology and Biotechnology, vol. 72, no. 6, pp. 1161-1169.
Canakci, M & Sanli, H 2008, 'Biodiesel production from various feedstocks and their effects on the fuel properties', Journal of Industrial Microbiology & Biotechnology, vol. 35, no. 5, pp. 431-441.
Carmona, ML, Naganuma, T & Yamaoka, Y 2003, 'Identification by HPLC-MS of carotenoids of the Thraustochytrium CHN-1 strain isolated from the Seto Inland Sea', Bioscience, Biotechnology and Biochemistry, vol. 67, no. 4, pp. 884-888.
Carrera, J, Baeza, JA, Vicent, T & Lafuente, J 2003, 'Biological nitrogen removal of high-strength ammonium industrial wastewater with two-sludge system', Water Research, vol. 37, no. 17, pp. 4211-4221.
Cavalier-Smith, T, Allsopp, MT & Chao, EE 1994, 'Thraustochytrids are chromists, not Fungi: 18s rRNA signatures of Heterokonta', Philosophical Transactions of the Royal Society B: Biological Sciences, vol. 346, no. 1318, pp. 387-397.
Certik, M & Shimizu, S 1999, 'Biosynthesis and regulation of microbial polyunsaturated fatty acid production', Journal of Bioscience and Bioengineering, vol. 87, no. 1, pp. 1-14.
Chang, WC, Lee, LP & Liepmann, D 2005, 'Biomimetic technique for adhesion-based collection and separation of cells in a microfluidic channel', Lab on a Chip, vol. 5, no. 1, pp. 64-73.
Chatdumrong, W, Yongmanitchai, W, Limtong, S & Worawattanamateekul, W 2007, 'Optimization of docosahexaenoic acid (DHA) production and improvement of astaxanthin content in a mutant Schizochytrium limacinum isolated from mangrove forest in Thailand', Kasetsart Journal - Natural Science, vol. 41, no. 2, pp. 324-334.
177
Chaung, K-C, Chu, C-Y, Su, Y-M & Che, Y-M 2012, 'Effect of culture conditions on growth, lipid content, and fatty acid composition of Aurantiochytrium mangrovei strain BL10', AMB Express, vol. 2, p. 42.
Chen, H, Chen, S-L & Jiang, J-G 2011, 'Effect of Ca2+ channel block on glycerol metabolism in Dunaliella salina under hypoosmotic and hyperosmotic Stresses', PLoS ONE, vol. 6, no. 12, p. e28613.
Chen, H, Li, D, Chen, J, Roberts, GJ, Saldeen, T & Mehta, JL 2003, 'EPA and DHA attenuate ox-LDL-induced expression of adhesion molecules in human coronary artery endothelial cells via protein kinase B pathway', Journal of Molecular and Cellular Cardiology, vol. 35, no. 7, pp. 769-775.
Chen, H, Zhang, Y, He, C & Wang, Q 2014, 'Ca2+ signal transduction related to neutral lipid synthesis in an oil-producing green alga Chlorella sp. C2', Plant Cell Physiol., vol. 55, no. 3, pp. 634-644.
Chen, W, Zhang, C, Song, L, Sommerfeld, M & Hu, Q 2009, 'A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae', Journal of Microbiological Methods, vol. 77, no. 1, pp. 41-47.
Chi, Z, Hu, B, Liu, Y, Frear, C, Wen, Z & Chen, S 2007a, 'Production of ω-3 polyunsaturated fatty acids from cull potato using an algae culture process', Applied Biochemistry and Biotechnology, vol. 137-140, no. 1-12, pp. 805-815.
Chi, Z, Liu, Y, Frear, C & Chen, S 2009, 'Study of a two-stage growth of DHA-producing marine algae Schizochytrium limacinum SR21 with shifting dissolved oxygen level', Applied Microbiology and Biotechnology, vol. 81, no. 6, pp. 1141-1148.
Chi, Z, Pyle, D, Wen, Z, Frear, C & Chen, S 2007b, 'A laboratory study of producing docosahexaenoic acid from biodiesel-waste glycerol by microalgal fermentation', Process Biochem, vol. 42, pp. 1537-1545.
Chisti, Y 2007, 'Biodiesel from microalgae', Biotechnology Advances, vol. 25, no. 3, pp. 294-306.
Chisti, Y 2013, 'Constraints to commercialization of algal fuels', Journal of Biotechnology, vol. 167, no. 3, pp. 201-214.
178
Cordero, BF, Obraztsova, I, Couso, I, Leon, R, Vargas, MA & Rodriguez, H 2011, 'Enhancement of lutein production in Chlorella sorokiniana (Chorophyta) by improvement of culture conditions and random mutagenesis', Marine Drugs, vol. 9, pp. 1607-1624.
Craik, JCA & Harvey, SM 1987, 'A biochemical method for distinguishing between wild and farmed salmonid fishes by their carotenoid pigmentation', Journal of the Forensic Science Society, vol. 27, no. 1, pp. 47-55.
Curtis, CL, Harwood, JL, Dent, CM & Caterson, B 2004, 'Biological basis for the benefit of nutraceutical supplementation in arthritis', Drug Discovery Today, vol. 9, no. 4, pp. 165-172.
Damare, V & Raghukumar, S 2008, 'Abundance of Thraustochytrids and bacteria in the equatorial Indian Ocean, in relation to transparent exopolymeric particles (TEPs)', FEMS Microbiology Ecology, vol. 65, no. 1, pp. 40-49.
De Swaaf, ME, de Rijk, TC, van der Meer, P, Eggink, G & Sijtsma, L 2003a, 'Analysis of docosahexaenoic acid biosynthesis in Crypthecodinium cohnii by 13C labelling and desaturase inhibitor experiments', Journal of Biotechnology, vol. 103, no. 1, pp. 21-29.
De Swaaf, ME, Sijtsma, L & Pronk, JT 2003b, 'High-cell-density fed-batch cultivation of the docosahexaenoic acid producing marine alga Crypthecodinium cohnii', Biotechnolology and Bioengineering, vol. 81, no. 6, pp. 666-672.
Doan, TTY, Sivaloganathan, B & Obbard, JP 2011, 'Screening of marine microalgae for biodiesel feedstock', Biomass and Bioenergy, vol. 35, no. 7, pp. 2534-2544.
Eler, GJ, Peralta, RM & Bracht, A 2009, 'The action of n-propyl gallate on gluconeogenesis and oxygen uptake in the rat liver', Chemico-Biological Interactions, vol. 181, no. 3, pp. 390-399.
Ethier, S, Woisard, K, Vaughan, D & Wen, Z 2011, 'Continuous culture of the microalgae Schizochytrium limacinum on biodiesel-derived crude glycerol for producing docosahexaenoic acid', Bioresour Technol, vol. 102, pp. 88-93.
Evans, CT & Ratledge, C 1984, 'Effect of nitrogen source on lipid accumulation in oleaginous yeasts', Journal of General Microbiology, vol. 130, no. 7, pp. 1693-1704.
179
Fan, K-W, Jiang, Y, Ho, L-T & Chen, F 2009, 'Differentiation in fatty acid profiles of pigmented and nonpigmented Aurantiochytrium isolated from Hong Kong Mangroves', Journal of Agricultural and Food Chemistry, vol. 57, no. 14, pp. 6334-6341.
Fan, KW, Jiang, Y, Faan, YW & Chen, F 2007, 'Lipid characterization of mangrove Thraustochytrid - Schizochytrium mangrovei', J Agric Food Chem, vol. 55, no. 8, pp. 2906-2910.
Fan, KW, Vrijmoed, LLP & Jones, EBG 2002a, 'Physiological studies of subtropical mangrove thraustochytrids', Botanica Marina, vol. 45, no. 1, pp. 50-57.
Fan, KW, Vrijmoed, LLP & Jones, EBG 2002b, 'Zoospore chemotaxis of mangrove Thraustochytrids from Hong Kong', Mycologia, vol. 94, no. 4, pp. 569-578.
Felsenstein J. 1985, 'Confidence limits on phylogenies: An approach using the bootstrap', Evolution, vol. 39, pp. 783-791.
Feng, F-Y, Yang, W, Jiang, G-Z, Xu, Y-N & Kuang, T-Y 2005, 'Enhancement of fatty acid production of Chlorella sp. (Chlorophyceae) by addition of glucose and sodium thiosulphate to culture medium', Process Biochemistry, vol. 40, no. 3–4, pp. 1315-1318.
Fenical, W & Jensen, PR 2006, 'Developing a new resource for drug discovery: marine actinomycete bacteria', Nat Chem Biol, vol. 2, no. 12, pp. 666-673.
Galafassi, S, Cucchetti, D, Pizza, F, Franzosi, G, Bianchi, D & Compagno, C 2012, 'Lipid production for second generation biodiesel by the oleaginous yeast Rhodotorula graminis', Bioresour Technolol, vol. 111, no. 0, pp. 398-403.
Ganuza, E, Anderson, AJ & Ratledge, C 2008, 'High-cell-density cultivation of Schizochytrium sp. in an ammonium/pH-auxostat fed-batch system', Biotechnology Letters, vol. 30, no. 9, pp. 1559-1564.
Gao-Qiang Liu, Q-LL, Xian-Chun Jin, Xiao-Ling Wang, Yan Zhao 2010, 'Screening and fermentation optimization of microbial lipid-producing molds from forest soils', African Journal of Microbiology Research, vol. 4, no. 14, pp. 1462-1468.
Garrill, A, Clipson, NJW & Jennings, DH 1992, 'Preliminary observations on the monovalent cation relations of Thraustochytrium aureum, a fungus requiring sodium for growth', Mycological Research, vol. 96, no. 4, pp. 295-304.
180
Gharieb, MM, Sayer, JA & Gadd, GM 1998, 'Solubilization of natural gypsum (CaSO4.2H2O) and the formation of calcium oxalate by Aspergillus niger and Serpula himantioides', Mycological Research, vol. 102, no. 7, pp. 825-830.
'The Global Market for EPA/DHA Omega-3 Products', Packaged Facts 2012, Packaged Facts, vol. http://www.packagedfacts.com/about/release.asp?id=3004.
Gu, Z, Deming, C, Yongbin, H, Zhigang, C & Feirong, G 2008, 'Optimization of carotenoids extraction from Rhodobacter sphaeroides', Lebenson Wiss Technol, vol. 41, pp. 1082-1088.
Guerin, M, Huntley, ME & Olaizola, M 2003, 'Haematococcus astaxanthin: applications for human health and nutrition', Trends Biotechnol, vol. 21, no. 5, pp. 210-216.
Guesnet, P & Alessandri, J-M 2011, 'Docosahexaenoic acid (DHA) and the developing central nervous system (CNS) – Implications for dietary recommendations', Biochimie, vol. 93, no. 1, pp. 7-12.
Gupta, A, Barrow, CJ & Puri, M 2012a, 'Omega-3 biotechnology: Thraustochytrids as a novel source of omega-3 oils', Biotechnol Adv, vol. 30, no. 6.
Gupta, A, Singh, D, Barrow, CJ & Puri, M 2013a, 'Exploring potential use of Australian Thraustochytrids for the bioconversion of glycerol to omega-3 and carotenoids production', Biochem Eng J, vol. 78, no. 0, pp. 11-17.
Gupta, A, Vongsvivut, J, Barrow, CJ & Puri, M 2012b, 'Molecular identification of marine yeast and its spectroscopic analysis establishes unsaturated fatty acid accumulation', J Biosci Bioeng, vol. 114, pp. 411-417.
Gupta, A, Wilkens, S, Adcock, J, Puri, M & Barrow, CJ 2013b, 'Pollen baiting facilitates the isolation of marine Thraustochytrids with potential in omega-3 and biodiesel production', Journal of Industrial Microbiology & Biotechnology, pp. 1-10.
Han, KH & Frazier, AB 2005, 'A microfluidic system for continuous magnetophoretic separation of suspended cells using their native magnetic properties', Proc. Nanotech, vol. 1, pp. 187-190.
Hauvermale, A, Kuner, J, Rosenzweig, B, Guerra, D, Diltz, S & Metz, JG 2006, 'Fatty acid production in Schizochytrium sp.: Involvement of a polyunsaturated fatty acid synthase and a type I fatty acid synthase', Lipids, vol. 41, no. 8, pp. 739-747.
181
Hayakawa, M, Tamura, T, Iino, H & Nonomura, H 1991, 'Pollen-baiting and drying method for the highly selective isolation of Actinoplanes spp. from soil', Journal of Fermentation and Bioengineering, vol. 72, no. 6, pp. 433-438.
Höller, U, Wright, AD, Matthee, GF, Konig, GM, Draeger, S, Aust, H-J & Schulz, B 2000, 'Fungi from marine sponges: diversity, biological activity and secondary metabolites', Mycological Research, vol. 104, no. 11, pp. 1354-1365.
Honda, D, Yokochi, T, Nakahara, T, Raghukumar, S, Nakagiri, A, Schaumann, K & Higashihara, T 1999, 'Molecular phylogeny of Labyrinthulids and Thraustochytrids based on the sequencing of 18S ribosomal RNA gene', Journal of Eukaryotic Microbiology, vol. 46, no. 6, pp. 637-647.
Hong, W-K, Yu, A, Heo, S-Y, Oh, B-R, Kim, C, Sohn, J-H, Yang, J-W, Kondo, A & Seo, J-W 2013, 'Production of lipids containing high levels of docosahexaenoic acid from empty palm fruit bunches by Aurantiochytrium sp. KRS101', Bioprocess and Biosystems Engineering, vol. 36, no. 7, pp. 959-963.
Hong, WK, Kim, CH, Rairakhwada, D, Kim, S, Hur, BK, Kondo, A & Seo, JW 2012, 'Growth of the oleaginous microalga Aurantiochytrium sp. KRS101 on cellulosic biomass and the production of lipids containing high levels of docosahexaenoic acid', Bioprocess and Biosystems Engineering, vol. 35, no. 1-2, pp. 129-133.
Hong, WK, Rairakhwada, D, Seo, PS, Park, SY, Hur, BK, Kim, CH & Seo, JW 2011, 'Production of lipids containing high levels of docosahexaenoic acid by a newly isolated microalga, Aurantiochytrium sp. KRS101', Applied Biochemistry and Biotechnology, vol. 164, no. 8, pp. 1468-1480.
Hu, C, Wu, S, Wang, Q, Jin, G, Shen, H & Zhao, Z 2011, 'Simultaneous utilization of glucose and xylose for lipid production by Trichosporon cutaneum', Biotechnol Biofuels, vol. 4, no. 1, p. 25.
Huang, J, Aki, T, Hachida, K, Yokochi, T, Kawamoto, S, Shigeta, S, Ono, K & Suzuki, O 2001, 'Profile of polyunsaturated fatty acids produced by Thraustochytrium sp. KK17-3', JAOCS, Journal of the American Oil Chemists' Society, vol. 78, no. 6, pp. 605-610.
Huang, J, Jiang, X, Zhang, X, Chen, W, Tian, B, Shu, Z & Hu, S 2008, 'Expressed sequence tag analysis of marine fungus Schizochytrium producing docosahexaenoic acid', Journal of Biotechnology, vol. 138, no. 1-2, pp. 9-16.
182
Huang, JZ, Jiang, XZ, Xia, XF, Yu, AQ, Mao, RY, Chen, XF & Tian, BY 2011, 'Cloning and functional identification of delta5 fatty acid desaturase gene and Its 5′-Upstream Region from Marine Fungus Thraustochytrium sp. FJN-10', Marine Biotechnology, vol. 13, no. 1, pp. 12-21.
Huang, TY, Lu, WC & Chu, IM 2012, 'A fermentation strategy for producing docosahexaenoic acid in Aurantiochytrium limacinum SR21 and increasing C22:6 proportions in total fatty acid', Bioresour Technol, vol. 123, no. 0, pp. 8-14.
Huang, Y, Zheng, H & Yan, Y 2010, 'Optimization of lipase-catalyzed transesterification of lard for biodiesel production using response surface methodology', Applied Biochemistry and Biotechnology, vol. 160, no. 2, pp. 504-515.
Iida, I, Nakahara, T, Yokochi, T, Kamisaka, Y, Yagi, H, Yamaoka, M & Suzuki, O 1996, 'Improvement of docosahexaenoic acid production in a culture of Thraustochytrium aureum by medium optimization', Journal of Fermentation and Bioengineering, vol. 81, no. 1, pp. 76-78.
'International Data Base, world population summary', 2010, US Census Bureau, vol. http://www.census.gov/ipc/www/idb/worldpopinfo.php.
Issa, AA 1996, 'The role of calcium in the stress response of the halotolerant green alga Dunaliella bardawil', Phyton (Horn, Austria), vol. 36, pp. 295-302.
Jakobsen, A, Aasen, I, Josefsen, K & Strøm, A 2008a, 'Accumulation of docosahexaenoic acid-rich lipid in Thraustochytrid Aurantiochytrium sp. strain T66: effects of N and P starvation and O2 limitation', Applied Microbiology and Biotechnology, vol. 80, no. 2, pp. 297-306.
Jakobsen, AN, Aasen, IM & Strøm, AR 2007, 'Endogenously synthesized (-)-proto-quercitol and glycine betaine are principal compatible solutes of Schizochytrium sp. strain S8 (ATCC 20889) and three new isolates of phylogenetically related thraustochytrids', Applied and Environmental Microbiology, vol. 73, no. 18, pp. 5848-5856.
Johnson, EJ 2002, 'The Role of Carotenoids in Human Health', Nutrition in Clinical Care, vol. 5, no. 2, pp. 56-65.
Johnson, MB & Wen, Z 2009, 'Production of biodiesel fuel from the microalga Schizochytrium limacinum by direct transesterification of algal biomass', Energy and Fuels, vol. 23, no. 10, pp. 5179-5183.
183
Jones, GM & O'Dor, RK 1983, 'Ultrastructural observations on a Thraustochytrid fungus parasitic in the gills of squid, Journal of Parasitology', Journal of Parasitology, vol. 69, no. 5, pp. 903-911.
Jørgensen, H 2009, 'Effect of nutrients on fermentation of pretreated wheat straw at very high dry matter content by Saccharomyces cerevisiae', Applied Biochemistry and Biotechnology, vol. 153, no. 1-3, pp. 44-57.
Jung, IS & Lovitt, RW 2010, 'Integrated production of long chain polyunsaturated fatty acids (PUFA)-rich Schizochytrium biomass using a nutrient supplemented marine aquaculture wastewater', Aquacultural Engineering, vol. 43, no. 2, pp. 51-61.
Kamble, A & Banerjee, UC 2008, 'Optimization of crucial reaction conditions for the production of nicotinamide by nitrile hydratase using response surface methodology', App Biochem and Biotechnol, vol. 151, no. 2-3, pp. 143-150.
Kanchana, R, Muraleedharan, UD & Raghukumar, S 2011, 'Alkaline lipase activity from the marine protists, Thraustochytrids', World Journal of Microbiology and Biotechnology, vol. 27, no. 9, pp. 2125-2131.
Kang, DH, Anbu, P, Jeong, YS, Chaulagain, BP, Seo, JW & Hur, BK 2010, 'Identification and characterization of a novel enzyme related to the synthesis of PUFAs derived from Thraustochytrium aureum ATCC 34304', Biotechnology and Bioprocess Engineering, vol. 15, no. 2, pp. 261-272.
Kang, DH, Anbu, P, Kim, WH & Hur, BK 2008, 'Coexpression of Elo-like enzyme and Δ5, Δ4-desaturases derived from Thraustochytrium aureum ATCC 34304 and the production of DHA and DPA in Pichia pastoris', Biotechnology and Bioprocess Engineering, vol. 13, no. 4, pp. 483-490.
Karakashev, D, Thomsen, A & Angelidaki, I 2007, 'Anaerobic biotechnological approaches for production of liquid energy carriers from biomass', Biotechnology Letters, vol. 29, no. 7, pp. 1005-1012.
Karhumaa, K, Sanchez, R, Hahn-Hägerdal, B & Gorwa-Grauslund, M-F 2007, 'Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae', Microbial Cell Factories, vol. 6, no. 1, pp. 1-10.
184
Kato, Y & Imamura, N 2008, 'Effect of calcium ion on uptake of amino acids by symbiotic Chlorella F36-ZK isolated from Japanese Paramecium bursaria', Plant Science, vol. 174, no. 1, pp. 88-96.
Kawashima, H, Akimoto, K, Shirasaka, N & Shimizu, S 1996, 'Inhibitory effects of alkyl gallate and its derivatives on fatty acid desaturation', Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, vol. 1299, no. 1, pp. 34-38.
Khozin-Goldberg, I, Bigogno, C & Cohen, Z 1999, 'Salicylhydroxamic acid inhibits Δ6 desaturation in the microalga Porphyridium cruentum', Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, vol. 1439, no. 3, pp. 384-394.
Khozin-Goldberg, I & Cohen, Z 2011, 'Unraveling algal lipid metabolism: Recent advances in gene identification', Biochimie, vol. 93, no. 1, pp. 91-100.
Kim, K, Jung Kim, E, Ryu, B-G, Park, S, Choi, Y-E & Yang, J-W 2013, 'A novel fed-batch process based on the biology of Aurantiochytrium sp. KRS101 for the production of biodiesel and docosahexaenoic acid', Bioresour Technol, vol. 135, no. 0, pp. 269-274.
Kimura, H, Fukuba, T & Naganuma, T 1999, 'Biomass of Thraustochytrid protoctists in coastal water', Marine Ecology Progress Series, vol. 189, pp. 27-33.
Kimura, M 1980, 'A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences', Journal of Molecular Evolution, vol. 16, no. 2, pp. 111-120.
Kobayashi, T, Sakaguchi, K, Matsuda, T, Abe, E, Hama, Y, Hayashi, M, Honda, D, Okita, Y, Sugimoto, S, Okino, N & Ito, M 2011, 'Increase of eicosapentaenoic acid in Thraustochytrids through Thraustochytrid ubiquitin promoter-driven expression of a fatty acid Δ5 desaturase gene', Applied and Environmental Microbiology, vol. 77, no. 11, pp. 3870-3876.
Kumon, Y, Yokochi, T & Nakahara, T 2005, 'High yield of long-chain polyunsaturated fatty acids by labyrinthulids on soybean lecithin-dispersed agar medium', Applied Microbiology and Biotechnology, vol. 69, no. 3, pp. 253-258.
Kumon, Y, Yokoyama, R, Haque, Z, Yokochi, T, Honda, D & Nakahara, T 2006, 'A new labyrinthulid isolate that produces only docosahexaenoic acid', Marine Biotechnology, vol. 8, no. Copyright (C) 2012 American Chemical Society (ACS). All Rights Reserved., pp. 170-177.
185
Kumon, Y, Yokoyama, R, Yokochi, T, Honda, D & Nakahara, T 2003, 'A new labyrinthulid isolate, which solely produces n-6 docosapentaenoic acid', Applied Microbiology and Biotechnology, vol. 63, no. 1, pp. 22-28.
Kumon, YK, Yokochi, TY, Nakahara, TN, Yamaoka, MY & Mito, KM 2002, 'Production of long-chain polyunsaturated fatty acids by monoxenic growth of labyrinthulids on oil-dispersed agar medium', Applied Microbiology and Biotechnology, vol. 60, no. 3, pp. 275-280.
Lam, MK & Lee, KT 2012, 'Microalgae biofuels: A critical review of issues, problems and the way forward', Biotechnology Advances, vol. 30, no. 3, pp. 673-690.
Leaño, EM, Gapasin, RSJ, Polohan, B & Vrijmoed, LLP 2003, 'Growth and fatty acid production of Thraustochytrids from Panay mangroves, Philippines', Fungal Diversity, vol. 12, pp. 111-122.
Lee Chang, K, Dumsday, G, Nichols, P, Dunstan, G, Blackburn, S & Koutoulis, A 2013, 'High cell density cultivation of a novel Aurantiochytrium sp. strain TC 20 in a fed-batch system using glycerol to produce feedstock for biodiesel and omega-3 oils', Applied Microbiology and Biotechnology, vol. 97, no. 15, pp. 6907-6918.
Lee Chang, KJ, Dunstan, GA, Abell, GCJ, Clementson, LA, Blackburn, SI, Nichols, PD & Koutoulis, A 2012, 'Biodiscovery of new Australian thraustochytrids for production of biodiesel and long-chain omega-3 oils', Applied Microbiology and Biotechnology, vol. 93, no. 5, pp. 2215-2231.
Lee Chang, KJ, Mansour, MP, Dunstan, GA, Blackburn, SI, Koutoulis, A & Nichols, PD 2011, 'Odd-chain polyunsaturated fatty acids in thraustochytrids', Phytochemistry, vol. 72, no. 11-12, pp. 1460-1465.
Lee, JC, Anbu, P, Kim, WH, Noh, MJ, Lee, SJ, Seo, JW & Hur, BK 2008, 'Identification of δ9-elongation activity from Thraustochytrium aureum by heterologous expression in Pichia pastoris', Biotechnology and Bioprocess Engineering, vol. 13, no. 5, pp. 524-532.
Lee, JY, Yoo, C, Jun, SY, Ahn, CY & Oh, HM 2010, 'Comparison of several methods for effective lipid extraction from microalgae', Bioresource Technology, vol. 101, pp. S75-S77.
Lee, M-H, Lee, C-H, Oh, T-K, Song, JK & Yoon, J-H 2006, 'Isolation and characterization of a novel lipase from a metagenomic library of tidal flat sediments: evidence for a new family of bacterial lipases', Applied and Environmental Microbiology, vol. 72, no. 11, pp. 7406-7409.
186
Lee, RA & Lavoie, J-M 2013, 'From first- to third-generation biofuels: Challenges of producing a commodity from a biomass of increasing complexity', Animal Frontiers, vol. 3, no. 2, pp. 6-11.
Lewis, TE, Nichols, PD & McMeekin, TA 2001, 'Sterol and squalene content of a docosahexaenoic-acid-producing Thraustochytrid: Influence of culture age, temperature, and dissolved oxygen', Marine Biotechnology, vol. 3, no. 5, pp. 439-447.
Li, M, Liu, G-L, Chi, Z & Chi, Z-M 2010, 'Single cell oil production from hydrolysate of cassava starch by marine-derived yeast Rhodotorula mucilaginosa TJY15a', Biomass and Bioenergy, vol. 34, no. 1, pp. 101-107.
Li, Q, Chen, GQ, Fan, KW, Lu, FUP, Aki, T & Jiang, Y 2009, 'Screening and characterization of squalene-producing thraustochytrids from Hong Kong mangroves', Journal of Agricultural and Food Chemistry, vol. 57, no. 10, pp. 4267-4272.
Li, Q, Du, W & Liu, D 2008, 'Perspectives of microbial oils for biodiesel production', Applied Microbiology and Biotechnology, vol. 80, no. 5, pp. 749-756.
Lian, M, Huang, H, Ren, L, Ji, X, Zhu, J & Jin, L 2010, 'Increase of docosahexaenoic acid production by Schizochytrium sp. through mutagenesis and enzyme assay', Applied Biochemistry and Biotechnology, vol. 162, no. 4, pp. 935-941.
Liang, Y, Sarkany, N, Cui, Y, Yesuf, J, Trushenski, J & Blackburn, JW 2010, 'Use of sweet sorghum juice for lipid production by Schizochytrium limacinum SR21', Bioresource Technology, vol. 101, no. 10, pp. 3623-3627.
Lin, L, Cunshan, Z, Vittayapadung, S, Xiangqian, S & Mingdong, D 2011, 'Opportunities and challenges for biodiesel fuel', Applied Energy, vol. 88, no. 4, pp. 1020-1031.
Lin, Y & Tanaka, S 2006, 'Ethanol fermentation from biomass resources: current state and prospects', Applied Microbiology and Biotechnology, vol. 69, no. 6, pp. 627-642.
Ling, X, Guo, J, Liu, X, Zhang, X, Wang, N, Lu, Y & Ng, IS 'Impact of carbon and nitrogen feeding strategy on high production of biomass and docosahexaenoic acid (DHA) by Schizochytrium sp. LU310', Bioresource Technology, no. 0.
Linko, YY & Hayakawa, K 1996, 'Docosahexaenoic acid: A valuable nutraceutical?', Trends in Food Science & Technology, vol. 7, no. 2, pp. 59-63.
187
Lippmeier, JC, Crawford, KS, Owen, CB, Rivas, AA, Metz, JG & Apt, KE 2009, 'Characterization of both polyunsaturated fatty acid biosynthetic pathways in Schizochytrium sp', Lipids, vol. 44, no. 7, pp. 621-630.
Mandal, S & Mallick, N 2009, 'Microalga Scenedesmus obliquus as a potential source for biodiesel production', Applied Microbiology and Biotechnology, vol. 84, no. 2, pp. 281-291.
Matsuda, T, Sakaguchi, K, Hamaguchi, R, Kobayashi, T, Abe, E, Hama, Y, Hayashi, M, Honda, D, Okita, Y, Sugimoto, S, Okino, N & Ito, M 2012, 'Analysis of Δ12-fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304', Journal of Lipid Research, vol. 53, no. 6, pp. 1210-1222.
Meher, LC, Vidya Sagar, D & Naik, SN 2006, 'Technical aspects of biodiesel production by transesterification—a review', Renewable and Sustainable Energy Reviews, vol. 10, no. 3, pp. 248-268.
Metz, JG, Kuner, J, Rosenzweig, B, Lippmeier, JC, Roessler, P & Zirkle, R 2009, 'Biochemical characterization of polyunsaturated fatty acid synthesis in Schizochytrium: Release of the products as free fatty acids', Plant Physiology and Biochemistry, vol. 47, no. 6, pp. 472-478.
Miao, X & Wu, Q 2006, 'Biodiesel production from heterotrophic microalgal oil', Bioresource Technology, vol. 97, no. 6, pp. 841-846.
Michelon, M, Matos de Borba, T, Silva Rafael, R, Burkert, C & Medeiros Burkert, J 2012, 'Extraction of carotenoids from Phaffia rhodozyma: A comparison between different techniques of cell disruption', Food Science and Biotechnology, vol. 21, pp. 1-8.
Minowa, T, Yokoyama, S-y, Kishimoto, M & Okakura, T 1995, 'Oil production from algal cells of Dunaliella tertiolecta by direct thermochemical liquefaction', Fuel, vol. 74, no. 12, pp. 1735-1738.
Mo, C, Douek, J & Rinkevich, B 2002, 'Development of a PCR strategy for Thraustochytrid identification based on 18S rDNA sequence', Marine Biology, vol. 140, no. 5, pp. 883-889.
Moellering, ER & Benning, C 2010, 'RNA interference silencing of a major lipid droplet protein affects lipid droplet size in Chlamydomonas reinhardtii', Eukaryotic Cell, vol. 26, no. 1-2, pp. 11-16.
188
Muryanto, S, Bayuseno, AP, Ma’mun, H, Usamah, M & Jotho 2014, 'Calcium Carbonate Scale Formation in Pipes: Effect of Flow Rates, Temperature, and Malic Acid as Additives on the Mass and Morphology of the Scale', Procedia Chemistry, vol. 9, no. 0, pp. 69-76.
Mutanda, T, Ramesh, D, Karthikeyan, S, Kumari, S, Anandraj, A & Bux, F 2011, 'Bioprospecting for hyper-lipid producing microalgal strains for sustainable biofuel production', Bioresource Technology, vol. 102, no. 1, pp. 57-70.
Nagano, N, Matsui, S, Kuramura, T, Taoka, Y, Honda, D & Hayashi, M 2011a, 'The distribution of extracellular cellulase activity in marine eukaryotes, Thraustochytrids', Marine Biotechnology, vol. 13, no. 2, pp. 133-136.
Nagano, N, Sakaguchi, K, Taoka, Y, Okita, Y, Honda, D, Ito, M & Hayashi, M 2011b, 'Detection of Genes Involved in Fatty Acid Elongation and Desaturation in Thraustochytrid Marine Eukaryotes', Journal of Oleo Science, vol. 60, no. 9, pp. 475-481.
Nagano, N, Taoka, Y, Honda, D & Hayashi, M 2009, 'Optimization of culture conditions for growth and docosahexaenoic acid production by a marine Thraustochytrid, Aurantiochytrium limacinum mh0186', Journal of Oleo Science, vol. 58, no. 12, pp. 623-628.
Nagaraj, S, Rajaram, MG, Arulmurugan, P, Baskaraboopathy, A, Karuppasamy, K, Jayappriyan, KR, Sundararaj, R & Rengasamy, R 2012, 'Antiproliferative potential of astaxanthin-rich alga Haematococcus pluvialis Flotow on human hepatic cancer (HepG2) cell line', Biomedicine & Preventive Nutrition, vol. 2, no. 3, pp. 149-153.
Nakagawa, Y, Moldéus, P & Moore, GA 1996, 'Relationship between mitochondrial dysfunction and toxicity of propyl gallate in isolated rat hepatocytes', Toxicology, vol. 114, no. 2, pp. 135-145.
Nakagawa, Y & Tayama, K 1998, 'Mechanism of mitochondrial dysfunction and cytotoxicity induced by tropolones in isolated rat hepatocytes', Chemico-Biological Interactions, vol. 116, no. 1–2, pp. 45-60.
Nakazawa, A, Matsuura, H, Kose, R, Kato, S, Honda, D, Inouye, I, Kaya, K & Watanabe, MM 2012, 'Optimization of culture conditions of the thraustochytrid Aurantiochytrium sp. strain 18W-13a for squalene production', Bioresource Technology, vol. 109, pp. 287-291.
Ngangkham, M, Ratha, S, Prasanna, R, Saxena, A, Dhar, D, Sarika, C & Prasad, R 2012, 'Biochemical modulation of growth, lipid quality and productivity in mixotrophic cultures of Chlorella sorokiniana', SpringerPlus, vol. 1, pp. 1 - 13.
189
Nie, Y, Liu, H, Du, G & Chen, J 2008, 'Acetate yield increased by gas circulation and fed-batch fermentation in a novel syntrophic acetogenesis and homoacetogenesis coupling system', Bioresource Technology, vol. 99, no. 8, pp. 2989-2995.
O’Grady, J & Morgan, J 2011, 'Heterotrophic growth and lipid production of Chlorella protothecoides on glycerol', Bioprocess and Biosystems Engineering, vol. 34, pp. 121-125.
Oclarit, JM & Belarmino, IBM 2009, 'Quantitative analysis of omega-3 aliphatic acid from Schizochytrium sp. DZAM (order Thraustochytriales division chromista) under different spectral photo-emissions', in Bremen.
Palmqvist, E, Grage, H, Meinander, NQ & Hahn-Hägerdal, B 1999, 'Main and interaction effects of acetic acid, furfural, and p-hydroxybenzoic acid on growth and ethanol productivity of yeasts', Biotechnol Bioeng, vol. 63, no. 1, pp. 46-55.
Palozza, P, Torelli, C, Boninsegna, A, Simone, R, Catalano, A, Mele, MC & Picci, N 2009, 'Growth-inhibitory effects of the astaxanthin-rich alga Haematococcus pluvialis in human colon cancer cells', Cancer Letters, vol. 283, no. 1, pp. 108-117.
Pereira, H, Barreira, L, Mozes, A, Florindo, C, Polo, C, Duarte, C, Custodio, L & Varela, J 2011, 'Microplate-based high throughput screening procedure for the isolation of lipid-rich marine microalgae', Biotechnology for Biofuels, vol. 4, p. 61.
Perez-Garcia, O, Escalante, FME, de-Bashan, LE & Bashan, Y 2011, 'Heterotrophic cultures of microalgae: Metabolism and potential products', Water Res., vol. 45, pp. 11-36.
Perveen, Z, Ando, H, Ueno, A, Ito, Y, Yamamoto, Y, Yamada, Y, Takagi, T, Kaneko, T, Kogame, K & Okuyama, H 2006, 'Isolation and characterization of a novel Thraustochytrid-like microorganism that efficiently produces docosahexaenoic acid', Biotechnology Letters, vol. 28, no. 3, pp. 197-202.
Poudyal, H, Panchal, SK, Ward, LC & Brown, L 2013, 'Effects of ALA, EPA and DHA in high-carbohydrate, high-fat diet-induced metabolic syndrome in rats', The Journal of Nutritional Biochemistry, vol. 24, no. 6, pp. 1041-1052.
Puri, M, Abraham, RE & Barrow, CJ 2012a, 'Biofuel production: Prospects, challenges and feedstock in Australia', Renewable and Sustainable Energy Reviews, vol. 16, no. 8, pp. 6022-6031.
190
Puri, M, Kaur, A, Singh, R & Singh, A 2010, 'Response surface optimization of medium components for naringinase production from Staphylococcus xylosus MAK2', Applied Biochemistry and Biotechnology, vol. 162, no. 1, pp. 181-191.
Puri, M, Sharma, D & Barrow, CJ 2012b, 'Enzyme-assisted extraction of bioactives from plants', Trends in biotechnology, vol. 30, no. 1, pp. 37-44.
Pyle, D & Wen, Z 2008a, 'Production of Omega-3 fatty acid-rich microalgae from biodiesel derived crude glycerol: Effects of glycerol impurities on algal growth and DHA production', in Providence, RI, vol. 3, pp. 1767-1778.
Pyle, DJ, Garcia, RA & Wen, Z 2008b, 'Producing docosahexaenoic acid (DHA)-rich algae from biodiesel-derived crude glycerol: Effects of impurities on DHA production and algal biomass composition', J Agric Food Chem, vol. 56, no. 11, pp. 3933-3939.
Qiu, X, Hong, H & MacKenzie, SL 2001, 'Identification of a Δ4 fatty acid desaturase from Thraustochytrium sp. involved in the biosynthesis of docosahexanoic acid by heterologous expression in Saccharomyces cerevisiae and Brassica juncea', Journal of Biological Chemistry, vol. 276, no. 34, pp. 31561-31566.
Qu, L, Ji, XJ, Ren, LJ, Nie, ZK, Feng, Y, Wu, WJ, Ouyang, PK & Huang, H 2011, 'Enhancement of docosahexaenoic acid production by Schizochytrium sp. using a two-stage oxygen supply control strategy based on oxygen transfer coefficient', Lett Appl Microbiol, vol. 52, no. 1, pp. 22-27.
Qu, L, Ren, L-J & Huang, H 2013, 'Scale-up of docosahexaenoic acid production in fed-batch fermentation by Schizochytrium sp. based on volumetric oxygen-transfer coefficient', Biochemical Engineering Journal, vol. 77, no. 0, pp. 82-87.
Querques, G & Souied, EH 2014, 'The role of omega-3 and micronutrients in age-related macular degeneration', Survey of Ophthalmology, vol. 59, no. 5.
Quilodrán, B, Hinzpeter, I, Hormazabal, E, Quiroz, A & Shene, C 2010, 'Docosahexaenoic acid (C22:6n-3, DHA) and astaxanthin production by Thraustochytriidae sp. AS4-A1 a native strain with high similitude to Ulkenia sp.: Evaluation of liquid residues from food industry as nutrient sources', Enzyme And Microbial Technology, vol. 47, no. 1-2, pp. 24-30.
191
Quilodrán, B, Hinzpeter, I, Quiroz, A & Shene, C 2009, 'Evaluation of liquid residues from beer and potato processing for the production of docosahexaenoic acid (C22:6n-3, DHA) by native Thraustochytrid strains', World J Microbiol Biotechnol, vol. 25, no. 12, pp. 2121-2128.
R.A. Andersen, MK 2005, 'Traditional microalgae isolation techniques, In: Algal Culturing Techniques ', Elsevier academic press New York, pp. 83-100.
Rabinowitz, C, Douek, J, Weisz, R, Shabtay, A & Rinkevich, B 2006, 'Isolation and characterization of four novel thraustochytrid strains from a colonial tunicate', Indian Journal of Marine Sciences, vol. 35, no. 4, pp. 341-350.
Raghukumar, S 2008, 'Thraustochytrid Marine Protists: Production of PUFAs and Other Emerging Technologies', Marine Biotechnology, vol. 10, no. 6, pp. 631-640.
Raghukumar, S & Schaumann, K 1993, 'An epifluorescence microscopy method for direct detection and enumeration of the fungilike marine protists, the thraustochytrids', Limnology & Oceanography, vol. 38, no. 1, pp. 182-187.
Ranjan, A, Patil, C & Moholkar, VS 2010, 'Mechanistic Assessment of Microalgal Lipid Extraction', Industrial & Engineering Chemistry Research, vol. 49, no. 6, pp. 2979-2985.
Ratledge, C 1991, 'Microorganisms for lipids', Acta Biotechnol, vol. 11, pp. 429 - 438.
Ratledge, C 2004, 'Fatty acid biosynthesis in microorganisms being used for Single Cell Oil production', Biochimie, vol. 86, no. 11, pp. 807-815.
Ratledge, C, Kanagachandran, K, Anderson, A, Grantham, D & Stephenson, J 2001, 'Production of docosahexaenoic acid by Crypthecodinium cohnii grown in a pH-auxostat culture with acetic acid as principal carbon source', Lipids, vol. 36, no. 11, pp. 1241-1246.
Ratledge, C & Wynn, JP 2002, 'The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms', in Advances in Applied Microbiology, Academic Press, vol. Volume 51, pp. 1-51.
Ren, H-Y, Liu, B-F, Ma, C, Zhao, L & Ren, N-Q 2013a, 'A new lipid-rich microalga Scenedesmus sp. strain R-16 isolated using Nile red staining: effects of carbon and nitrogen sources and initial pH on the biomass and lipid production', Biotechnol Biofuels, vol. 6, no. 1, p. 143.
192
Ren, L-J, Feng, Y, Li, J, Qu, L & Huang, H 2013b, 'Impact of phosphate concentration on docosahexaenoic acid production and related enzyme activities in fermentation of Schizochytrium sp', Bioprocess and Biosystems Engineering, vol. 36, no. 9, pp. 1177-1183.
Ren, LJ, Huang, H, Xiao, AH, Lian, M, Jin, LJ & Ji, XJ 2009, 'Enhanced docosahexaenoic acid production by reinforcing acetyl-CoA and NADPH supply in Schizochytrium sp. HX-308', Bioprocess and Biosystems Engineering, vol. 32, no. 6, pp. 837-843.
Ren, LJ, Ji, XJ, Huang, H, Qu, L, Feng, Y, Tong, QQ & Ouyang, PK 2010, 'Development of a stepwise aeration control strategy for efficient docosahexaenoic acid production by Schizochytrium sp', Applied Microbiology and Biotechnology, vol. 87, no. 5, pp. 1649-1656.
Rodolfi, L, Chini Zittelli, G, Bassi, N, Padovani, G, Biondi, N, Bonini, G & Tredici, MR 2009, 'Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor', Biotechnology and Bioengineering, vol. 102, no. 1, pp. 100-112.
Rosa, SM, Galvagno, MA & Vélez, CG 2011, 'Adjusting culture conditions to isolate thraustochytrids from temperate and cold environments in southern Argentina', Mycoscience, vol. 52, no. 4, pp. 242-252.
Saenge, C, Cheirsilp, B, Suksaroge, TT & Bourtoom, T 2011, 'Potential use of oleaginous red yeast Rhodotorula glutinis for the bioconversion of crude glycerol from biodiesel plant to lipids and carotenoids', Process Biochem, vol. 46, no. 1, pp. 210-218.
Saitou, N & Nei, M 1987, 'The neighbor-joining method: a new method for reconstructing phylogenetic trees', Molecular Biology and Evolution, vol. 4, no. 4, pp. 406-425.
Sanil Kumar, V, Pathak, KC, Pednekar, P, Raju, NSN & Gowthaman, R 2006, 'Coastal processes along the Indian Coastline', Current science, vol. 91, no. 4, pp. 530-536.
Santocono, M, Zurria, M, Berrettini, M, Fedeli, D & Falcioni, G 2007, 'Lutein, zeaxanthin and astaxanthin protect against DNA damage in SK-N-SH human neuroblastoma cells induced by reactive nitrogen species', Journal of Photochemistry and Photobiology B: Biology, vol. 88, no. 1, pp. 1-10.
Sarada, R, Vidhyavathi, R, Usha, D & Ravishankar, GA 2006, 'An efficient method for extraction of astaxanthin from green alga Haematococcus pluvialis', Journal of Agriculture and Food Chemistry, vol. 54, pp. 7585-7588.
193
Scaife, MA, Burja, AM & Wright, PC 2009, 'Characterization of cyanobacterial β-carotene ketolase and hydroxylase genes in Escherichia coli, and their application for astaxanthin biosynthesis', Biotechnology and Bioengineering, vol. 103, no. 5, pp. 944-955.
Scorletti, E, Bhatia, L, McCormick, KG, Clough, GF, Nash, K, Calder, PC & Byrne, CD 2014, 'Design and rationale of the WELCOME trial: A randomised, placebo controlled study to test the efficacy of purified long chain omega-3 fatty treatment in non-alcoholic fatty liver disease', Contemporary Clinical Trials, vol. 37, no. 2, pp. 301-311.
Scott, SD, Armenta, RE, Berryman, KT & Norman, AW 2011, 'Use of raw glycerol to produce oil rich in polyunsaturated fatty acids by a Thraustochytrid', Enzyme Microb Technol, vol. 48, pp. 267-272.
Sedmak, JJ, Weerasinghe, D & Jolly, S 1990, 'Extraction and quantitation of astaxanthin from Phaffia rhodozyma', Biotechnology Techniques, vol. 4, pp. 107-112.
Shabala, L, McMeekin, T & Shabala, S 2009, 'Osmotic adjustment and requirement for sodium in marine protist thraustochytrid', Environmental Microbiology, vol. 11, no. 7, pp. 1835-1843.
Sharma, YC, Singh, B & Korstad, J 2011, 'A critical review on recent methods used for economically viable and eco-friendly development of microalgae as a potential feedstock for synthesis of biodiesel', Green Chemistry, vol. 13, no. 11, pp. 2993-3006.
Shen, Y, Pei, Z, Yuan, W & Moa, E 2009, 'Effect of nitrogen and extraction method on algae lipid yield', International Journal of Agriculture and Biological Engineering, vol. 2, pp. 51-57.
Shimizu, S, Jareonkitmongkol, S, Kawashima, H, Akimoto, K & Yamada, H 1992, 'Inhibitory effect of curcumin on fatty acid desaturation in Mortierella alpina 1S-4 and rat liver microsomes', Lipids, vol. 27, no. 7, pp. 509-512.
Shoresh, M, Spivak, M & Bernstein, N 2011, 'Involvement of calcium-mediated effects on ROS metabolism in the regulation of growth improvement under salinity', Free Radical Biology and Medicine, vol. 51, no. 6, pp. 1221-1234.
Singh, A & Ward, OP 1996, 'Production of high yields of docosahexaenoic acid by Thraustochytrium roseum ATCC 28210', J Ind Microbiol., vol. 16, no. 6, pp. 370-373.
194
Singh, A & Ward, OP 1997, 'Microbial Production of docosahexaenoic acid (DHA, C22:6)', in LN Saul & IL Allen (eds), Adv Appl Microbiol, Academic Press, vol. Volume 45, pp. 271-312.
Singh, D, Puri, M, Wilkens, S, Mathur, AS, Tuli, DK & Barrow, CJ 2013, 'Characterization of a new zeaxanthin producing strain of Chlorella saccharophila isolated from New Zealand marine waters', Bioresour Technol, vol. 143, no. 0, pp. 308-314.
Sinigalliano, CD, Winshell, J, Guerrero, MA, Scorzetti, G, Fell, JW, Eaton, RW, Brand, L & Rein, KS 2009, 'Viable Cell Sorting of Dinoflagellates by Multiparametric Flow Cytometry', Phycologia, vol. 48, no. 4, pp. 249-257.
Skender, B, Hofmanová, J, Slavík, J, Jelínková, I, Machala, M, Moyer, MP, Kozubík, A & Hyršlová Vaculová, A 2014, 'DHA-mediated enhancement of TRAIL-induced apoptosis in colon cancer cells is associated with engagement of mitochondria and specific alterations in sphingolipid metabolism', Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, vol. 1841, no. 9, pp. 1308-1317.
Snyder, CL, Yurchenko, OP, Siloto, RMP, Chen, X, Liu, Q, Mietkiewska, E & Weselake, RJ 2009, 'Acyltransferase action in the modification of seed oil biosynthesis', New Biotechnology, vol. 26, no. 1–2, pp. 11-16.
Somashekar, D & Joseph, R 2000, 'Inverse relationship between carotenoid and lipid formation in Rhodotorula gracilis according to the C/N ratio of the growth medium', World Journal of Microbiology and Biotechnology, vol. 16, no. 5, pp. 491-493.
Song, X, Zhang, X, Guo, N, Zhu, L & Kuang, C 2007a, 'Assessment of marine thraustochytrid Schizochytrium limacinum OUC88 for mariculture by enriched feeds', Fisher Science, vol. 73, no. 3, pp. 565-573.
Song, X, Zhang, X, Kuang, C, Zhu, L & Guo, N 2007b, 'Optimization of fermentation parameters for the biomass and DHA production of Schizochytrium limacinum OUC88 using response surface methodology', Process Biochemistry, vol. 42, no. 10, pp. 1391-1397.
Stephens, E, Ross, IL, Mussgnug, JH, Wagner, LD, Borowitzka, MA, Posten, C, Kruse, O & Hankamer, B 2010, 'Future prospects of microalgal biofuel production systems', Trends in Plant Science, vol. 15, no. 10, pp. 554-564.
Subramaniam, R, Dufreche, S, Zappi, M & Bajpai, R 2010, 'Microbial lipids from renewable resources: production and characterization', Journal of Industrial Microbiology & Biotechnology, vol. 37, no. 12, pp. 1271-1287.
195
Taha, A, Kimoto, T, Kanada, T & Okuyama, H 2013, 'Growth optimization of thraustochytrid strain 12B for the commercial production of docosahexaenoic acid', Food Science and Biotechnology, vol. 22, no. 1, pp. 53-58.
Takahashi, K, Hattori, A, Suzuki, I, Ichiki, T & Yasuda, K 2004, 'Non-destructive on chip cell sorting system with real-time microscopic image processing', J Nanobiotechnology, vol. 48, no. 4, pp. 249-257.
Takao, Y, Nagasaki, K, Mise, K, Okuno, T & Honda, D 2005, 'Isolation and characterization of a novel single-stranded RNA virus infectious to a marine fungoid protist, Schizochytrium sp. (Thraustochytriaceae, Labyrinthulea)', Applied and Environmental Microbiology, vol. 71, no. 8, pp. 4516-4522.
Takao, Y, Tomaru, Y, Nagasaki, K, Sasakura, Y, Yokoyama, R & Honda, D 2007, 'Fluorescence in situ hybridization using 18S rRNA-targeted probe for specific detection of thraustochytrids (Labyrinthulomycetes)', Plankton and Benthos Research, vol. 2, no. 2, pp. 91-97.
Talebnia, F, Karakashev, D & Angelidaki, I 2010, 'Production of bioethanol from wheat straw: An overview on pretreatment, hydrolysis and fermentation', Bioresour Technol, vol. 101, no. 13, pp. 4744-4753.
Tamura, K, Stecher, G, Peterson, D, Filipski, A & Kumar, S 2013, 'MEGA6: Molecular evolutionary genetics analysis version 6.0.', Molecular Biology and Evolution, vol. 30, pp. 2725-2729.
Taoka, Y, Nagano, N, Okita, Y, Izumida, H, Sugimoto, S & Hayashi, M 2008, 'Effect of addition of Tween 80 and potassium dihydrogenphosphate to basal medium on the isolation of marine eukaryotes, thraustochytrids', Journal of Bioscience and Bioengineering, vol. 105, no. 5, pp. 562-565.
Taoka, Y, Nagano, N, Okita, Y, Izumida, H, Sugimoto, S & Hayashi, M 2009, 'Extracellular enzymes produced by marine eukaryotes, Thraustochytrids', Bioscience, Biotechnology and Biochemistry, vol. 73, no. 1, pp. 180-182.
Taoka, Y, Nagano, N, Okita, Y, Izumida, H, Sugimoto, S & Hayashi, M 2010, 'Use of an antifungal drug, amphotericin B for isolation of Thraustochytrids', Journal of Bioscience and Bioengineering, vol. 110, no. 6, pp. 720-723.
196
Taoka, Y, Nagano, N, Okita, Y, Izumida, H, Sugimoto, S & Hayashi, M 2011, 'Effect of Tween 80 on the growth, lipid accumulation and fatty acid composition of Thraustochytrium aureum ATCC 34304', Journal of Bioscience and Bioengineering, vol. 111, no. 4, pp. 420-424.
Tapia V, E, Anschau, A, Coradini, AL, T Franco, T & Deckmann, A 2012, 'Optimization of lipid production by the oleaginous yeast Lipomyces starkeyi by random mutagenesis coupled to cerulenin screening', AMB Express, vol. 2, no. 1, p. 64.
Teir, S, Eloneva, S, Fogelholm, C-J & Zevenhoven, R 2007, 'Dissolution of steelmaking slags in acetic acid for precipitated calcium carbonate production', Energy, vol. 32, no. 4, pp. 528-539.
Unagul, P, Assantachai, C, Phadungruengluij, S, Pongsuteeragul, T, Suphantharika, M & Verduyn, C 2006, 'Biomass and docosahexaenoic acid formation by Schizochytrium mangrovei Sk-02 at low salt concentrations', Botanica Marina, vol. 49, no. 2, pp. 182-190.
Unagul, P, Assantachai, C, Phadungruengluij, S, Suphantharika, M, Tanticharoen, M & Verduyn, C 2007, 'Coconut water as a medium additive for the production of docosahexaenoic acid (C22:6 n3) by Schizochytrium mangrovei Sk-02', Bioresour Technol, vol. 98, no. 2, pp. 281-287.
Valduga, E, Valério, A, Tatsch, P, Treichel, H, Furigo, A, Jr. & Luccio, M 2009, 'Assessment of cell disruption and carotenoids extraction from Sporidiobolus salmonicolor (CBS 2636)', Food and Bioprocess Technology, vol. 2, pp. 234-238.
Wall, R, Ross, RP, Fitzgerald, GF & Stanton, C 2010, 'Fatty acids from fish: the anti-inflammatory potential of long-chain omega-3 fatty acids', Nutrition Reviews, vol. 68, no. 5, pp. 280-289.
Ward, OP & Singh, A 2005, 'Omega-3/6 fatty acids: Alternative sources of production', Process Biochemistry, vol. 40, no. 12, pp. 3627-3652.
Wilkens, SL & Maas, EW 2012, 'Development of a novel technique for axenic isolation and culture of thraustochytrids from New Zealand marine environments', Journal of Applied Microbiology, vol. 112, no. 2, pp. 346-352.
William, B, Craig, W & James, M 2005, 'Development of a docosahexaenoic acid production technology using Schizochytrium', in Single Cell Oils, AOCS Publishing.
197
Wong, MKM, Tsui, CKM, Au, DWT & Vrijmoed, LLP 2008, 'Docosahexaenoic acid production and ultrastructure of the Thraustochytrid Aurantiochytrium mangrovei MP2 under high glucose concentrations', Mycoscience, vol. 49, no. 4, pp. 266-270.
Wu, ST, Yu, ST & Lin, LP 2005, 'Effect of culture conditions on docosahexaenoic acid production by Schizochytrium sp. S31', Process Biochemistry, vol. 40, no. 9, pp. 3103-3108.
Wynn, JP, Hamid, AA & Ratledge, C 1999, 'The role of malic enzyme in the regulation of lipids accumulation in filamentous fungi', Microbiology, vol. 145, no. 8, pp. 1911-1917.
Xu, J, Du, W, Zhao, X, Zhang, G & Liu, D 2013, 'Microbial oil production from various carbon sources and its use for biodiesel preparation', Biofuels, Bioproducts and Biorefining, vol. 7, no. 1, pp. 65-77.
Yaguchi, T, Tanaka, S, Yokochi, T, Nakahara, T & Higashihara, T 1997, 'Production of high yields of docosahexaenoic acid by Schizochytrium sp. strain SR21', JAOCS, Journal of the American Oil Chemists' Society, vol. 74, no. 11, pp. 1431-1434.
Yamasaki, T, Aki, T, Shinozaki, M, Taguchi, M, Kawamoto, S & Ono, K 2006, 'Utilization of Shochu distillery wastewater for production of polyunsaturated fatty acids and xanthophylls using Thraustochytrid', Journal of Bioscience and Bioengineering, vol. 102, no. 4, pp. 323-327.
Yan, J, Cheng, R, Lin, X, You, S, Li, K, Rong, H & Ma, Y 2013, 'Overexpression of acetyl-CoA synthetase increased the biomass and fatty acid proportion in microalga Schizochytrium', Applied Microbiology and Biotechnology, vol. 97, no. 5, pp. 1933-1939.
Yang, G, Liu, Y & Gao, M 2011, Effects of pH and aeration on the production of docosahexaenoic acid by Thraustochytrium aureum in controlled batch fermentor cultures, Harbin, 10226680 (ISSN); 9783037850220 (ISBN).
Yang, HL, Lu, CK, Chen, SF & Chen, YM 2010, 'Isolation and characterization of Taiwanese heterotrophic microalgae: Screening of strains for docosahexaenoic acid (DHA) production', Marine Biotechnology, vol. 12, no. 2, pp. 173-185.
Ye, J, Feng, L, Xiong, J & Xiong, Y 2011, 'Ultrasound-assisted extraction of corn carotenoids in ethanol', International Journal of Food Science & Technology, vol. 46, no. 10, pp. 2131-2136.
198
Yen, H-W, Yang, Y-C & Yu, Y-H 2012, 'Using crude glycerol and thin stillage for the production of microbial lipids through the cultivation of Rhodotorula glutinis', J Biosci Bioeng, vol. 114, no. 4, pp. 453-456.
Yokochi, T, Honda, D, Higashihara, T & Nakahara, T 1998, 'Optimization of docosahexaenoic acid production by Schizochytrium limacinum SR21', Applied Microbiology and Biotechnology, vol. 49, no. 1, pp. 72-76.
Yousuf, A 2012, 'Biodiesel from lignocellulosic biomass – Prospects and challenges', Waste Management, vol. 32, no. 11, pp. 2061-2067.
Yu, X, Zheng, Y, Dorgan, KM & Chen, S 2011, 'Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid', Bioresour Technol, vol. 102, no. 10, pp. 6134-6140.
Yu, Y, Wu, Y, Patch, C, Wu, Z, Szabo, A, Li, D & Huang, X-F 2013, 'DHA prevents altered 5-HT1A, 5-HT2A, CB1 and GABAA receptor binding densities in the brain of male rats fed a high-saturated-fat diet', The Journal of Nutritional Biochemistry, vol. 24, no. 7, pp. 1349-1358.
Zhang, G, French, WT, Hernandez, R, Alley, E & Paraschivescu, M 2011, 'Effects of furfural and acetic acid on growth and lipid production from glucose and xylose by Rhodotorula glutinis', Biomass Bioenergy, vol. 35, no. 1, pp. 734-740.
Zhao, X, Peng, F, Du, W, Liu, C & Liu, D 2012, 'Effects of some inhibitors on the growth and lipid accumulation of oleaginous yeast Rhodosporidium toruloides and preparation of biodiesel by enzymatic transesterification of the lipid', Bioprocess and Biosystems Engineering, vol. 35, no. 6, pp. 993-1004.
Zheng, Y, Chi, Z, Ahring, BK & Chen, S 2012, 'Oleaginous yeast Cryptococcus curvatus for biofuel production: Ammonia’s effect', Biomass Bioenergy, vol. 37, no. 0, pp. 114-121.
Zhu, L, Zhang, X, Ren, X & Zhu, Q 2008, 'Effects of culture conditions on growth and docosahexaenoic acid production from Schizochytrium limacinum', Journal of Ocean University of China, vol. 7, no. 1, pp. 83-88.